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We claim: 1. A method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a neonatal cell or a population of neonatal cells, comprising administering to the neonatal cell or the population of neonatal cells: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. 2. A method of expressing a polypeptide of interest from a target genomic locus in a neonatal cell or a population of neonatal cells, comprising administering to the neonatal cell or the population of neonatal cells: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus. 3. The method of claim 1 or 2, wherein the neonatal cell is a liver cell or the population of neonatal cells is a population of liver cells. 4. The method of any preceding claim, wherein the neonatal cell is a hepatocyte or the population of neonatal cells is a population of hepatocytes. 5. The method of any preceding claim, wherein the neonatal cell is a human cell or the population of neonatal cells is a population of human cells. 6. The method of claim 5, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 52 weeks after birth. 7. The method of claim 5, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 24 weeks after birth. 8. The method of claim 5, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 12 weeks after birth. 9. The method of claim 5, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 8 weeks after birth. 10. The method of claim 5, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 4 weeks after birth. 11. The method of any preceding claim, wherein the neonatal cell is in vitro or ex vivo or the population of neonatal cells is in vitro or ex vivo. 12. The method of any one of claims 1-10, wherein the neonatal cell is in vivo in a neonatal subject or the population of neonatal cells is in vivo in a neonatal subject. 13. A method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a neonatal cell in a neonatal subject, comprising administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. 14. A method of expressing a polypeptide of interest from a target genomic locus in a neonatal cell in a neonatal subject, comprising administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus. 15. A method of expressing a polypeptide of interest from a target genomic locus in a neonatal cell in a neonatal subject, comprising administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, wherein the subject comprises a mutation in a genome in the subject, wherein the mutation results in reduced activity or expression of an endogenous polypeptide having enzymatic activity. 16. The method of claim 15, wherein the nucleic acid encoding the polypeptide of interest encodes a polypeptide having the enzymatic activity of a wild type polypeptide encoded by the gene in which the subject has a mutation that results in reduced activity or expression of the endogenous polypeptide. 17. The method of any one of claims 13-16, wherein the neonatal cell is a liver cell. 18. The method of any one of claims 13-17, wherein the neonatal cell is a hepatocyte. 19. The method of any one of claims 13-18, wherein the neonatal cell is a human cell. 20. A method of treating an enzyme deficiency in a neonatal subject in need thereof, comprising administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the polypeptide of interest comprises an enzyme to treat the enzyme deficiency; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the enzyme deficiency. 21. A method of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a neonatal subject in need thereof, comprising administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the lysosomal storage disease is characterized by a loss-of-function of the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the enzyme deficiency. 22. The method of claim 20 or 21, wherein the neonatal subject has a disease of a bleeding disorder characterized by the enzyme deficiency. 23. The method of claim 22, wherein the bleeding disorder is selected from hemophilia A, hemophilia B, and von Willebrand disease. 24. The method of claim 20 or 21, wherein the neonatal subject has a disease of an inborn error of metabolism characterized by the enzyme deficiency. 25. The method of claim 20 or 21, wherein the neonatal subject has a disease selected from Krabbe disease (galactosylceramidase), phenylketonuria, galactosemia, maple syrup urine disease, mitochondrial disorders, Friedreich ataxia, Zellweger syndrome, adrenoleukodystrophy, Wilson disease, hemochromatosis, ornithine transcarbamylase deficiency, methylmalonic academia, propionic academia, argininosuccinic aciduria, methylmalonic aciduria, type I citrullinemia/argininosuccinate synthetase deficiency, carbamoyl-phosphate synthase 1 deficiency, propionic acidemia, isovaleric acidemia, glutaric academia I, and progressive familial intrahepatic cholestasis, types 2 and 3, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Niemann-Pick disease type A, Niemann- Pick disease type BGM1-gangliosidosis, Sandhoff disease, Tay-Sachs disease, GM2- activator deficiency, GM3-gangliosidosis, metachromatic leukodystrophy, sphingolipid-activator deficiency, Scheie disease, Hurler-Scheie disease, Hurler disease, Hunter disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Morquio syndrome A, Morquio syndrome B, Maroteaux-Lamy disease, Sly disease, MPS IX, or Pompe disease. 26. The method of claim 20 or 21, wherein the neonatal subject has a lysosomal storage disease characterized by the enzyme deficiency. 27. A method of treating a lysosomal storage disease in a neonatal subject in need thereof, comprising administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the lysosomal storage disease is characterized by a loss-of-function of the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the lysosomal storage disease. 28. A method of preventing or reducing the onset of a sign or symptom of a lysosomal storage disease in a neonatal subject in need thereof, comprising administering to the neonatal subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the lysosomal storage disease is characterized by a loss-of-function of the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the lysosomal storage disease. 29. The method of any one of claims 12-28, wherein the neonatal subject is a human subject. 30. The method of claim 29, wherein the neonatal subject is within 52 weeks after birth. 31. The method of claim 29, wherein the neonatal subject is within 24 weeks after birth. 32. The method of claim 29, wherein the neonatal subject is within 12 weeks after birth. 33. The method of claim 29, wherein the neonatal subject is within 8 weeks after birth. 34. The method of claim 29, wherein the neonatal subject is within 4 weeks after birth. 35. The method of any one of claims 12-34, wherein the method results in increased expression of the polypeptide of interest in the subject compared to a method comprising administering an episomal expression vector encoding the polypeptide of interest to a control subject. 36. The method of any one of claims 12-35, wherein the method results in increased serum levels of the polypeptide of interest in the subject compared to a method comprising administering an episomal expression vector encoding the polypeptide of interest to a control subject. 37. The method of any one of claims 12-36, wherein the expression or activity of the polypeptide of interest is at least 50% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at six months after the administering. 38. The method of any one of claims 12-37, wherein the expression or activity of the polypeptide of interest is at least 50% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at one year after the administering. 39. The method of any one of claims 12-38, wherein the expression or activity of the polypeptide of interest is at least 60% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at six months after the administering. 40. The method of any one of claims 12-39, wherein expression or activity of the polypeptide of interest is at least 50% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at two years after the administering. 41. The method of any one of claims 12-40, wherein the expression or activity of the polypeptide of interest is at least 60% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at two years after the administering. 42. The method of any one of claims 12-41, wherein the expression or activity of the polypeptide of interest is at least 60% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at six months after the administering. 43. The method of any one of claim 12-42, wherein the method further comprises assessing preexisting AAV immunity in the neonatal subject prior to administering the nucleic acid construct to the subject. 44. The method of claim 43, wherein the preexisting AAV immunity is preexisting AAV8 immunity. 45. The method of claim 43 or 44, wherein assessing preexisting AAV immunity comprises assessing immunogenicity using a total antibody immune assay or a neutralizing antibody assay. 46. The method of any preceding claim, wherein the nucleic acid construct is administered simultaneously with the nuclease agent or the one or more nucleic acids encoding the nuclease agent. 47. The method of any one of claims 1-45, wherein the nucleic acid construct is not administered simultaneously with the nuclease agent or the one or more nucleic acids encoding the nuclease agent. 48. The method of claim 47, wherein the nucleic acid construct is administered prior to the nuclease agent or the one or more nucleic acids encoding the nuclease agent. 49. The method of claim 47, wherein the nucleic acid construct is administered after the nuclease agent or the one or more nucleic acids encoding the nuclease agent. 50. The method of any preceding claim, wherein the polypeptide of interest comprises a therapeutic polypeptide. 51. The method of any preceding claim, wherein the polypeptide of interest is a secreted polypeptide. 52. The method of claim 50 or 51, wherein the polypeptide of interest comprises a hydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, saposin-C activator, ceramidase, sphingomyelinase, β-hexosaminidase, GM2 activator, GM3 synthase, arylsulfatase, sphingolipid activator, α-iduronidase, iduronidase-2-sulfatase, heparin N-sulfatase, N-acetyl-α-glucosaminidase, α-glucosamide N-acetyltransferase, N-acetylglucosamine-6- sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, N-acetylgalactosamine-4-sulfatase, β- glucuronidase, or a hyaluronidase. 53. The method of claim 52, wherein the polypeptide of interest comprises lysosomal alpha-glucosidase. 54. The method of any preceding claim, wherein the polypeptide of interest comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 173, optionally wherein the polypeptide of interest is encoded by a codon-optimized and CpG-depleted nucleotide sequence. 55. The method of any preceding claim, wherein the coding sequence for the polypeptide of interest comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence selected from SEQ ID NOS: 174-182 and 581-588, optionally selected from SEQ ID NOS: 175-179, wherein the nucleotide sequence is codon-optimized and CpG-depleted. 56 The method of any preceding claim, wherein the nucleic acid construct is CpG depleted. 57. The method of any one of claims 52-56, wherein the polypeptide of interest comprises a delivery domain. 58. The method of claim 57, wherein the polypeptide of interest is delivered to and internalized by skeletal muscle and heart tissue in the subject. 59. The method of any preceding claim, wherein the subject has an infantile- onset genetic disorder. 60. The method of any preceding claim, wherein the subject wherein the subject has Pompe disease. 61. The method of any one of claims 1-51, wherein the subject has a bleeding disorder. 62. The method of claim 61, wherein the polypeptide of interest is Factor VIII, Factor IX, or von Willebrand factor. 63. The method of any one of claims 1-50, wherein the polypeptide of interest is an intracellular polypeptide. 64. The method of any preceding claim, wherein the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the polypeptide of interest. 65. The method of any preceding claim, wherein the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the polypeptide of interest. 66. The method of any one of claims 1-63, wherein the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the polypeptide of interest, and the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the polypeptide of interest. 67. The method of any preceding claim, wherein the nucleic acid construct does not comprise a homology arm. 68. The method of claim 67, wherein the nucleic acid construct is inserted into the target genomic locus via non-homologous end joining. 69. The method of any one of claims 1-66, wherein the nucleic acid construct comprises homology arms. 70. The method of claim 69, wherein the nucleic acid construct is inserted into the target genomic locus via homology-directed repair. 71. The method of any preceding claim, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the polypeptide of interest. 72. The method of any preceding claim, wherein the nucleic acid construct is single-stranded DNA or double-stranded DNA. 73. The method of claim 72, wherein the nucleic acid construct is single- stranded DNA. 74. The method of any preceding claim, wherein the nucleic acid construct is a bidirectional nucleic acid construct. 75. The method of claim 74, wherein the nucleic acid construct comprises: (I) a first segment comprising the coding sequence for the polypeptide of interest; and (II) a second segment comprising a reverse complement of a second coding sequence for the polypeptide of interest. 76. The method of claim 75, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the coding sequence for the polypeptide of interest, a first polyadenylation signal or sequence, a reverse complement of a second polyadenylation signal or sequence, the reverse complement of the second coding sequence for the polypeptide of interest, and a reverse complement of a second splice acceptor. 77. The method of claim 75 or 76, wherein the coding sequence for the polypeptide of interest and the second coding sequence for the polypeptide of interest are different. 78. The method of any preceding claim, wherein the nucleic acid construct is in a nucleic acid vector or a lipid nanoparticle. 79. The method of claim 78, wherein the nucleic acid construct is in the nucleic acid vector. 80. The method of claim 79, wherein the nucleic acid vector is a viral vector. 81. The method of claim 79 or 80, wherein the nucleic acid vector is an adeno-associated viral (AAV) vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 160, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 160. 82. The method of claim 81, wherein the AAV vector is a single-stranded AAV (ssAAV) vector. 83. The method of claim 81 or 82, wherein the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector. 84. The method of claim 83, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector. 85. The method of claim 84, wherein the AAV vector is a single-stranded rAAV8 vector. 86. The method of any preceding claim, wherein the nucleic acid construct is CpG-depleted. 87. The method of any preceding claim, wherein the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene. 88. The method of claim 87, wherein the nuclease target site is in intron 1 of the albumin gene. 89. The method of any preceding claim, wherein the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. 90. The method of any one of claims 1-89, wherein the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. 91. The method of claim 90, wherein the guide RNA target sequence is in intron 1 of an albumin gene. 92. The method of claim 91, wherein the albumin gene is a human albumin gene. 93. The method of any one of claims 90-92, wherein: (I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 30-61, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 36, 30, 33, and 41; and/or (II) the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 30-61, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 36, 30, 33, and 41. 94. The method of any one of claims 90-93, wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 30-61, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 36, 30, 33, and 41. 95. The method of any one of claims 90-94, wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 30-61, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 36, 30, 33, and 41. 96. The method of any one of claims 90-95, wherein the guide RNA comprises any one of SEQ ID NOS: 62-125, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 68, 100, 62, 94, 65, 97, 73, and 105. 97. The method of any one of claims 90-96, wherein: (I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 36; and/or (II) the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 36. 98. The method of any one of claims 90-97, wherein the DNA-targeting segment comprises SEQ ID NO: 36. 99. The method of any one of claims 90-98, wherein the DNA-targeting segment consists of SEQ ID NO: 36. 100. The method of any one of claims 90-99, wherein the guide RNA comprises SEQ ID NO: 68 or 100. 101. The method of any one of claims 90-100, wherein the method comprises administering the guide RNA in the form of RNA. 102. The method of any one of claims 90-101, wherein the guide RNA comprises at least one modification. 103. The method of claim 102, wherein the at least one modification comprises a 2’-O-methyl-modified nucleotide. 104. The method of claim 102 or 103, wherein the at least one modification comprises a phosphorothioate bond between nucleotides. 105. The method of any one of claims 102-104, wherein the at least one modification comprises a modification at one or more of the first five nucleotides at the 5’ end of the guide RNA. 106. The method of any one of claims 102-105, wherein the at least one modification comprises a modification at one or more of the last five nucleotides at the 3’ end of the guide RNA. 107. The method of any one of claims 102-106, wherein the at least one modification comprises phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA. 108. The method of any one of claims 102-107, wherein the at least one modification comprises phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA. 109. The method of any one of claims 102-108, wherein the at least one modification comprises 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA. 110. The method of any one of claims 102-109, wherein the at least one modification comprises 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. 111. The method of any one of claims 102-110, wherein the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. 112. The method of any one of claims 90-111, wherein the guide RNA is a single guide RNA (sgRNA). 113. The method of any one of claims 90-112, wherein the method comprises administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 100, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. 114. The method of any one of claims 90-113, wherein the Cas protein is a Cas9 protein. 115. The method of claim 114, wherein the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Campylobacter jejuni Cas9 protein, a Streptococcus thermophilus Cas9 protein, or a Neisseria meningitidis Cas9 protein. 116. The method of claim 114, wherein the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. 117. The method of any one of claims 90-116, wherein the Cas protein comprises the sequence set forth in SEQ ID NO: 11. 118. The method of any one of claims 90-117, wherein the nucleic acid encoding the Cas protein is codon-optimized for expression in a mammalian cell or a human cell. 119. The method of any one of claims 90-118, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. 120. The method of claim 119, wherein the mRNA encoding the Cas protein comprises at least one modification. 121. The method of claim 119 or 120, wherein the mRNA encoding the Cas protein is modified to comprise a modified uridine at one or more or all uridine positions. 122. The method of claim 121, wherein the modified uridine is pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. 123. The method of claim 121 or 122, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1- methyl-pseudouridine. 124. The method of any one of claims 119-123, wherein the mRNA encoding the Cas protein comprises a 5’ cap. 125. The method of any one of claims 119-124, wherein the mRNA encoding the Cas protein comprises a polyadenylation sequence. 126. The method of any one of claims 119-125, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 2, 1, or 12. 127. The method of any one of claims 90-126, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 2, 1, or 12, and the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a polyadenylation sequence. 128. The method of any one of claims 90-127, wherein the method comprises administering the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 68 or 100, and wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 2, 1, or 12. 129. The method of any one of claims 90-128, wherein the method comprises administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 100, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 2, 1, or 12, and the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl- pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a polyadenylation sequence. 130. The method of any one of claims 90-129, wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. 131. The method of claim 130, wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. 132. The method of claim 131, wherein the cationic lipid is Lipid A ((9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate). 133. The method of claim 130 or 131, wherein the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 134. The method of any one of claims 131-133, wherein the helper lipid is cholesterol. 135. The method of any one of claims 131-134, wherein the stealth lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG). 136. The method of any one of claims 131-135, wherein the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k- DMG. 137. The method of any one of claims 131-136, wherein the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. 138. The method of any one of claims 90-137, wherein the albumin gene is a human albumin gene, wherein the method comprises administering the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 68 or 100, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 2, 1, or 12, and wherein the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. 139. The method of any one of claims 90-137, wherein the albumin gene is a human albumin gene, wherein the method comprises administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 100, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl- modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O- methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 2, 1, or 12, and the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl- pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a polyadenylation sequence, and wherein the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. 140. A neonatal cell or a population of neonatal cells made by the method of any preceding claim. 141. A neonatal cell or a population of neonatal cells comprising a nucleic acid construct comprising a coding sequence for a polypeptide of interest inserted into a target genomic locus. 142. The neonatal cell or the population of neonatal cells of claim 140 or 141, wherein the neonatal cell is a liver cell or the population of neonatal cells is a population of liver cells. 143. A cell or a population of cells made by the method of any one of claims 1- 139. 144. A cell or a population of cells comprising a nucleic acid construct comprising a coding sequence for a polypeptide of interest inserted into a target genomic locus. 145. The cell or the population of cells of claim 143 or 144, wherein the cell is a liver cell or the population of cells is a population of liver cells. 146. The neonatal cell or the population of neonatal cells of any one of claims 140-142 or the cell or population of cells of any one of claims 143-145, wherein the cell or the neonatal cell is a hepatocyte or the population of cells or the population of neonatal cells is a population of hepatocytes. 147. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146 or the cell or population of cells of any one of claims 143-146, wherein the cell or the neonatal cell is a human cell or the population of cells or the population of neonatal cells is a population of human cells. 148. The neonatal cell or the population of neonatal cells of any one of claims 140-142, 146, and 147, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 52 weeks after birth. 149. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-148, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 24 weeks after birth. 150. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-149, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 12 weeks after birth. 151. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-150, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 8 weeks after birth. 152. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-151, wherein the neonatal cell or the population of neonatal cells is from a neonatal subject within 4 weeks after birth. 153. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-152 or the cell or population of cells of any one of claims 143-147, wherein the cell or the neonatal cell is in vitro or ex vivo or the population of cells or the population of neonatal cells is in vitro or ex vivo. 154. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-152 or the cell or population of cells of any one of claims 143-147, wherein the cell or the neonatal cell is in vivo in a subject or the population of cells or the population of neonatal cells is in vivo. 155. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-154 or the cell or population of cells of any one of claims 143-147 and 153- 154, wherein the polypeptide of interest is expressed. 156. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-155 or the cell or population of cells of any one of claims 143-147 and 153- 155, wherein the polypeptide of interest comprises a therapeutic polypeptide, optionally wherein the polypeptide of interest comprises lysosomal alpha-glucosidase. 157. The neonatal cell or population of neonatal cells of claim 140-142 and 146-156 or the cell or population of cells of any one of claims 143-147 and 153-156, wherein the lysosomal alpha-glucosidase comprises the amino acid sequence of SEQ ID NO: 173, optionally wherein the polypeptide of interest is encoded by a nucleic acid is codon-optimized and CpG- depleted nucleotide sequence. 158. The neonatal cell or population of neonatal cells of claim 156 or 157 or the cell or population of cells of claim 156 or 157, wherein the lysosomal alpha-glucosidase is encoded by a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence selected from SEQ ID NOS: 174-182 and 581-588, optionally SEQ ID NOS: 175-179, wherein the nucleotide sequence is codon-optimized and CpG-depleted. 159. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-156 or the cell or population of cells of any one of claims 143-147 and 153- 156, wherein the polypeptide of interest is a secreted polypeptide. 160. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-156 or the cell or population of cells of any one of claims 143-147 and 153- 156, wherein the polypeptide of interest is an intracellular polypeptide. 161. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-160 or the cell or population of cells of any one of claims 143-147 and 153- 160, wherein the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the polypeptide of interest. 162. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-161 or the cell or population of cells of any one of claims 143-147 and 153- 161, wherein the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the polypeptide of interest. 163. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-160 or the cell or population of cells of any one of claims 143-147 and 153- 160, wherein the nucleic acid construct comprises a splice acceptor upstream of the coding sequence for the polypeptide of interest, and the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence for the polypeptide of interest. 164. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-163 or the cell or population of cells of any one of claims 143-147 and 153- 163, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the polypeptide of interest, and wherein the coding sequence for the polypeptide of interest is operably linked to an endogenous promoter at the target genomic locus. 165. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-164 or the cell or population of cells of any one of claims 143-147 and 153- 164, wherein the nucleic acid construct is a bidirectional nucleic acid construct. 166. The neonatal cell or the population of neonatal cells of claim 165 or the cell or the population of cells of claim 165, wherein the nucleic acid construct comprises: (I) a first segment comprising the coding sequence for the polypeptide of interest; and (II) a second segment comprising a reverse complement of a second coding sequence for the polypeptide of interest. 167. The neonatal cell or the population of neonatal cells of claim 166 or the cell or the population of cells of claim 166, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the coding sequence for the polypeptide of interest, a first polyadenylation signal or sequence, a reverse complement of a second polyadenylation signal or sequence, the reverse complement of the second coding sequence for the polypeptide of interest, and a reverse complement of a second splice acceptor. 168. The neonatal cell or the population of neonatal cells of claim 166 or 167 or the cell or the population of cells of claim 166 or 167, wherein the coding sequence for the polypeptide of interest and the second coding sequence for the polypeptide of interest are different. 169. The neonatal cell or the population of neonatal cells of any one of claims 140-142 and 146-168 or the cell or population of cells of any one of claims 143-147 and 153- 168, wherein the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene, optionally wherein the nuclease target site is in intron 1 of the albumin gene. |
[00261] Table 2. Human ALB Intron 1 Guide Sequences. [00262] Table 3. Human ALB Intron 1 sgRNA Sequences.
[00263] Table 4. Mouse Alb Intron 1 Guide Sequences. [00264] Table 5. Mouse Alb Intron 1 sgRNA Sequences. [00265] TracrRNAs can be in any form (e.g., full-length tracrRNAs or active partial tracrRNAs) and of varying lengths. They can include primary transcripts or processed forms. For example, tracrRNAs (as part of a single-guide RNA or as a separate molecule as part of a two- molecule gRNA) may comprise, consist essentially of, or consist of all or a portion of a wild type tracrRNA sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild type tracrRNA sequence). Examples of wild type tracrRNA sequences from S. pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide, and 65-nucleotide versions. See, e.g., Deltcheva et al. (2011) Nature 471(7340):602-607; WO 2014/093661, each of which is herein incorporated by reference in its entirety for all purposes. Examples of tracrRNAs within single-guide RNAs (sgRNAs) include the tracrRNA segments found within +48, +54, +67, and +85 versions of sgRNAs, where “+n” indicates that up to the +n nucleotide of wild type tracrRNA is included in the sgRNA. See US 8,697,359, herein incorporated by reference in its entirety for all purposes. [00266] The percent complementarity between the DNA-targeting segment of the guide RNA and the complementary strand of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%). The percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be at least 60% over about 20 contiguous nucleotides. As an example, the percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be 100% over the 14 contiguous nucleotides at the 5’ end of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting segment can be considered to be 14 nucleotides in length. As another example, the percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be 100% over the seven contiguous nucleotides at the 5’ end of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting segment can be considered to be 7 nucleotides in length. In some guide RNAs, at least 17 nucleotides within the DNA-targeting segment are complementary to the complementary strand of the target DNA. For example, the DNA-targeting segment can be 20 nucleotides in length and can comprise 1, 2, or 3 mismatches with the complementary strand of the target DNA. In one example, the mismatches are not adjacent to the region of the complementary strand corresponding to the protospacer adjacent motif (PAM) sequence (i.e., the reverse complement of the PAM sequence) (e.g., the mismatches are in the 5’ end of the DNA- targeting segment of the guide RNA, or the mismatches are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the region of the complementary strand corresponding to the PAM sequence). [00267] The protein-binding segment of a gRNA can comprise two stretches of nucleotides that are complementary to one another. The complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein-binding segment of a subject gRNA interacts with a Cas protein, and the gRNA directs the bound Cas protein to a specific nucleotide sequence within target DNA via the DNA-targeting segment. [00268] Single-guide RNAs can comprise a DNA-targeting segment and a scaffold sequence (i.e., the protein-binding or Cas-binding sequence of the guide RNA). For example, such guide RNAs can have a 5’ DNA-targeting segment joined to a 3’ scaffold sequence. Exemplary scaffold sequences (e.g., for use with S. pyogenes Cas9) comprise, consist essentially of, or consist of: or (version 8; SEQ ID NO: 28). In some guide sgRNAs, the four terminal U residues of version 6 are not present. In some sgRNAs, only 1, 2, or 3 of the four terminal U residues of version 6 are present. Guide RNAs targeting any of the guide RNA target sequences disclosed herein can include, for example, a DNA-targeting segment on the 5’ end of the guide RNA fused to any of the exemplary guide RNA scaffold sequences on the 3’ end of the guide RNA. That is, any of the DNA-targeting segments disclosed herein can be joined to the 5’ end of any one of the above scaffold sequences to form a single guide RNA (chimeric guide RNA). [00269] Guide RNAs can include modifications or sequences that provide for additional desirable features (e.g., modified or regulated stability; subcellular targeting; tracking with a fluorescent label; a binding site for a protein or protein complex; and the like). That is, guide RNAs can include one or more modified nucleosides or nucleotides, or one or more non- naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. Examples of such modifications include, for example, a 5’ cap (e.g., a 7-methylguanylate cap (m7G)); a 3’ polyadenylated tail (i.e., a 3’ poly(A) tail); a riboswitch sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and/or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin); a modification or sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, and so forth); a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like); and combinations thereof. Other examples of modifications include engineered stem loop duplex structures, engineered bulge regions, engineered hairpins 3’ of the stem loop duplex structure, or any combination thereof. See, e.g., US 2015/0376586, herein incorporated by reference in its entirety for all purposes. A bulge can be an unpaired region of nucleotides within the duplex made up of the crRNA-like region and the minimum tracrRNA- like region. A bulge can comprise, on one side of the duplex, an unpaired 5^-XXXY-3^ where X is any purine and Y can be a nucleotide that can form a wobble pair with a nucleotide on the opposite strand, and an unpaired nucleotide region on the other side of the duplex. [00270] Guide RNAs can comprise modified nucleosides and modified nucleotides including, for example, one or more of the following: (1) alteration or replacement of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (2) alteration or replacement of a constituent of the ribose sugar such as alteration or replacement of the 2’ hydroxyl on the ribose sugar (an exemplary sugar modification); (3) replacement (e.g., wholesale replacement) of the phosphate moiety with dephospho linkers (an exemplary backbone modification); (4) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (5) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (6) modification of the 3’ end or 5’ end of the oligonucleotide (e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker (such 3’ or 5’ cap modifications may comprise a sugar and/or backbone modification); and (7) modification or replacement of the sugar (an exemplary sugar modification). Other possible guide RNA modifications include modifications of or replacement of uracils or poly-uracil tracts. See, e.g., WO 2015/048577 and US 2016/0237455, each of which is herein incorporated by reference in its entirety for all purposes. Similar modifications can be made to Cas-encoding nucleic acids, such as Cas mRNAs. For example, Cas mRNAs can be modified by depletion of uridine using synonymous codons. [00271] Chemical modifications such at hose listed above can be combined to provide modified gRNAs and/or mRNAs comprising residues (nucleosides and nucleotides) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In one example, every base of a gRNA is modified (e.g., all bases have a modified phosphate group, such as a phosphorothioate group). For example, all or substantially all of the phosphate groups of a gRNA can be replaced with phosphorothioate groups. Alternatively or additionally, a modified gRNA can comprise at least one modified residue at or near the 5’ end. Alternatively or additionally, a modified gRNA can comprise at least one modified residue at or near the 3’ end. [00272] Some gRNAs comprise one, two, three or more modified residues. For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the positions in a modified gRNA can be modified nucleosides or nucleotides. [00273] Unmodified nucleic acids can be prone to degradation. Exogenous nucleic acids can also induce an innate immune response. Modifications can help introduce stability and reduce immunogenicity. Some gRNAs described herein can contain one or more modified nucleosides or nucleotides to introduce stability toward intracellular or serum-based nucleases. Some modified gRNAs described herein can exhibit a reduced innate immune response when introduced into a population of cells. [00274] The gRNAs disclosed herein can comprise a backbone modification in which the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. The modification can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. Backbone modifications of the phosphate backbone can also include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. [00275] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (Rp) or the “S” configuration (Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. [00276] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. [00277] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. [00278] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group (a sugar modification). For example, the 2’ hydroxyl group (OH) can be modified (e.g., replaced with a number of different oxy or deoxy substituents. Modifications to the 2’ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2’-alkoxide ion. [00279] Examples of 2’ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). The 2’ hydroxyl group modification can be 2’-O-Me. Likewise, the 2’ hydroxyl group modification can be a 2’-fluoro modification, which replaces the 2’ hydroxyl group with a fluoride. The 2’ hydroxyl group modification can include locked nucleic acids (LNA) in which the 2’ hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH 2 ) n -amino, (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). The 2’ hydroxyl group modification can include unlocked nucleic acids (UNA) in which the ribose ring lacks the C2’-C3’ bond. The 2’ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH 2 CH 2 OCH 3 , e.g., a PEG derivative). [00280] Deoxy 2’ modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2- amino (wherein amino can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein. [00281] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form (e.g. L- nucleosides). [00282] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base. [00283] In a dual guide RNA, each of the crRNA and the tracrRNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracrRNA. In a sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, and/or internal nucleosides may be modified, and/or the entire sgRNA may be chemically modified. Some gRNAs comprise a 5’ end modification. Some gRNAs comprise a 3’ end modification. [00284] The guide RNAs disclosed herein can comprise one of the modification patterns disclosed in WO 2018/107028 A1, herein incorporated by reference in its entirety for all purposes. The guide RNAs disclosed herein can also comprise one of the structures/modification patterns disclosed in US 2017/0114334, herein incorporated by reference in its entirety for all purposes. The guide RNAs disclosed herein can also comprise one of the structures/modification patterns disclosed in WO 2017/136794, WO 2017/004279, US 2018/0187186, or US 2019/0048338, each of which is herein incorporated by reference in its entirety for all purposes. [00285] As one example, nucleotides at the 5’ or 3’ end of a guide RNA can include phosphorothioate linkages (e.g., the bases can have a modified phosphate group that is a phosphorothioate group). For example, a guide RNA can include phosphorothioate linkages between the 2, 3, or 4 terminal nucleotides at the 5’ or 3’ end of the guide RNA. As another example, nucleotides at the 5’ and/or 3’ end of a guide RNA can have 2’-O-methyl modifications. For example, a guide RNA can include 2’-O-methyl modifications at the 2, 3, or 4 terminal nucleotides at the 5’ and/or 3’ end of the guide RNA (e.g., the 5’ end). See, e.g., WO 2017/173054 A1 and Finn et al. (2018) Cell Rep.22(9):2227-2235, each of which is herein incorporated by reference in its entirety for all purposes. Other possible modifications are described in more detail elsewhere herein. In a specific example, a guide RNA includes 2’-O- methyl analogs and 3’ phosphorothioate internucleotide linkages at the first three 5’ and 3’ terminal RNA residues. Such chemical modifications can, for example, provide greater stability and protection from exonucleases to guide RNAs, allowing them to persist within cells for longer than unmodified guide RNAs. Such chemical modifications can also, for example, protect against innate intracellular immune responses that can actively degrade RNA or trigger immune cascades that lead to cell death. [00286] As one example, any of the guide RNAs described herein can comprise at least one modification. In one example, the at least one modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide, a phosphorothioate (PS) bond between nucleotides, a 2’-fluoro (2’-F) modified nucleotide, or a combination thereof. For example, the at least one modification can comprise a 2’-O-methyl (2’-O-Me) modified nucleotide. Alternatively or additionally, the at least one modification can comprise a phosphorothioate (PS) bond between nucleotides. Alternatively or additionally, the at least one modification can comprise a 2’-fluoro (2’-F) modified nucleotide. In one example, a guide RNA described herein comprises one or more 2’- O-methyl (2’-O-Me) modified nucleotides and one or more phosphorothioate (PS) bonds between nucleotides. [00287] The modifications can occur anywhere in the guide RNA. As one example, the guide RNA comprises a modification at one or more of the first five nucleotides at the 5’ end of the guide RNA, the guide RNA comprises a modification at one or more of the last five nucleotides of the 3’ end of the guide RNA, or a combination thereof. For example, the guide RNA can comprise phosphorothioate bonds between the first four nucleotides of the guide RNA, phosphorothioate bonds between the last four nucleotides of the guide RNA, or a combination thereof. Alternatively or additionally, the guide RNA can comprise 2’-O-Me modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA, can comprise 2’-O-Me modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, or a combination thereof. [00288] In one example, a modified gRNA can comprise the following sequence: mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmA mGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 29), where “N” may be any natural or non-natural nucleotide. For example, the totality of N residues comprise a human ALB intron 1 DNA-targeting segment as described herein (e.g., the sequence set forth in SEQ ID NO: 29, wherein the N residues are replaced with the DNA- targeting segment of any one of SEQ ID NOS: 30-61, the DNA-targeting segment of any one of SEQ ID NOS: 36, 30, 33, and 41, or the DNA-targeting segment of SEQ ID NO: 36. For example, a modified gRNA can comprise the sequence set forth in any one of SEQ ID NOS: 94- 125, the sequence set forth in any one of SEQ ID NOS: 100, 94, 97, and 105, or the sequence set forth in SEQ ID NO: 100 in Table 3. The terms “mA,” “mC,” “mU,” and “mG” denote a nucleotide (A, C, U, and G, respectively) that has been modified with 2’-O-Me. The symbol depicts a phosphorothioate modification. In certain embodiments, A, C, G, U, and N independently denote a ribose sugar, i.e., 2’-OH. In certain embodiments in the context of a modified sequence, A, C, G, U, and N denote a ribose sugar, i.e., 2’-OH. A phosphorothioate linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos. The terms A*, C*, U*, or G* denote a nucleotide that is linked to the next (e.g., 3’) nucleotide with a phosphorothioate bond. The terms “mA*,” “mC*,” “mU*,” and “mG*” denote a nucleotide (A, C, U, and G, respectively) that has been substituted with 2’-O- Me and that is linked to the next (e.g., 3’) nucleotide with a phosphorothioate bond. [00289] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2’-fluoro (2’-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability. Abasic nucleotides refer to those which lack nitrogenous bases. Inverted bases refer to those with linkages that are inverted from the normal 5’ to 3' linkage (i.e., either a 5’ to 5’ linkage or a 3’ to 3’ linkage). [00290] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5’ nucleotide via a 5’ to 5’ linkage, or an abasic nucleotide may be attached to the terminal 3’ nucleotide via a 3’ to 3’ linkage. An inverted abasic nucleotide at either the terminal 5’ or 3’ nucleotide may also be called an inverted abasic end cap. [00291] In one example, one or more of the first three, four, or five nucleotides at the 5’ terminus, and one or more of the last three, four, or five nucleotides at the 3’ terminus are modified. The modification can be, for example, a 2’-O-Me, 2’-F, inverted abasic nucleotide, phosphorothioate bond, or other nucleotide modification well known to increase stability and/or performance. [00292] In another example, the first four nucleotides at the 5’ terminus, and the last four nucleotides at the 3’ terminus can be linked with phosphorothioate bonds. [00293] In another example, the first three nucleotides at the 5’ terminus, and the last three nucleotides at the 3’ terminus can comprise a 2’-O-methyl (2’-O-Me) modified nucleotide. In another example, the first three nucleotides at the 5’ terminus, and the last three nucleotides at the 3’ terminus comprise a 2’-fluoro (2’-F) modified nucleotide. In another example, the first three nucleotides at the 5’ terminus, and the last three nucleotides at the 3’ terminus comprise an inverted abasic nucleotide. [00294] Guide RNAs can be provided in any form. For example, the gRNA can be provided in the form of RNA, either as two molecules (separate crRNA and tracrRNA) or as one molecule (sgRNA), and optionally in the form of a complex with a Cas protein. The gRNA can also be provided in the form of DNA encoding the gRNA. The DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or separate RNA molecules (e.g., separate crRNA and tracrRNA). In the latter case, the DNA encoding the gRNA can be provided as one DNA molecule or as separate DNA molecules encoding the crRNA and tracrRNA, respectively. [00295] When a gRNA is provided in the form of DNA, the gRNA can be transiently, conditionally, or constitutively expressed in the cell. DNAs encoding gRNAs can be stably integrated into the genome of the cell and operably linked to a promoter active in the cell. Alternatively, DNAs encoding gRNAs can be operably linked to a promoter in an expression construct. For example, the DNA encoding the gRNA can be in a vector comprising a heterologous nucleic acid, such as a nucleic acid encoding a Cas protein. Alternatively, it can be in a vector or a plasmid that is separate from the vector comprising the nucleic acid encoding the Cas protein. Promoters that can be used in such expression constructs include promoters active, for example, in one or more of a eukaryotic cell, a human cell, a non-human cell, a mammalian cell, a non-human mammalian cell, a rodent cell, a mouse cell, a rat cell, a pluripotent cell, an embryonic stem (ES) cell, an adult stem cell, a developmentally restricted progenitor cell, an induced pluripotent stem (iPS) cell, or a one-cell stage embryo. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Such promoters can also be, for example, bidirectional promoters. Specific examples of suitable promoters include an RNA polymerase III promoter, such as a human U6 promoter, a rat U6 polymerase III promoter, or a mouse U6 polymerase III promoter. [00296] Alternatively, gRNAs can be prepared by various other methods. For example, gRNAs can be prepared by in vitro transcription using, for example, T7 RNA polymerase (see, e.g., WO 2014/089290 and WO 2014/065596, each of which is herein incorporated by reference in its entirety for all purposes). Guide RNAs can also be a synthetically produced molecule prepared by chemical synthesis. For example, a guide RNA can be chemically synthesized to include 2’-O-methyl analogs and 3’ phosphorothioate internucleotide linkages at the first three 5’ and 3’ terminal RNA residues. [00297] Guide RNAs (or nucleic acids encoding guide RNAs) can be in compositions comprising one or more guide RNAs (e.g., 1, 2, 3, 4, or more guide RNAs) and a carrier increasing the stability of the guide RNA (e.g., prolonging the period under given conditions of storage (e.g., -20°C, 4°C, or ambient temperature) for which degradation products remain below a threshold, such below 0.5% by weight of the starting nucleic acid or protein; or increasing the stability in vivo). Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, and lipid microtubules. Such compositions can further comprise a Cas protein, such as a Cas9 protein, or a nucleic acid encoding a Cas protein. [00298] As one example, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of the sequence set forth in any one of SEQ ID NOS: 62-125. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the DNA-targeting segment set forth in any one of SEQ ID NOS: 62-125. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 90% or at least 95% identical to the DNA-targeting segment set forth in any one of SEQ ID NOS: 62-125. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that differs by no more than 3, no more than 2, or no more than 1 nucleotide from the sequence set forth in any one of SEQ ID NOS: 62-125. [00299] As another example, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of the sequence set forth in any one of SEQ ID NOS: 68, 100, 62, 94, 65, 97, 73, and 105. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the DNA-targeting segment set forth in any one of SEQ ID NOS: 68, 100, 62, 94, 65, 97, 73, and 105. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 90% or at least 95% identical to the DNA-targeting segment set forth in any one of SEQ ID NOS: 68, 100, 62, 94, 65, 97, 73, and 105. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that differs by no more than 3, no more than 2, or no more than 1 nucleotide from the sequence set forth in any one of SEQ ID NOS: 68, 100, 62, 94, 65, 97, 73, and 105. [00300] As another example, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 68 or 100. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the DNA-targeting segment set forth in SEQ ID NO: 68 or 100. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 90% or at least 95% identical to the DNA- targeting segment set forth in SEQ ID NO: 68 or 100. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that differs by no more than 3, no more than 2, or no more than 1 nucleotide from the sequence set forth in SEQ ID NO: 68 or 100. [00301] As another example, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 62 or 94. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the DNA-targeting segment set forth in SEQ ID NO: 62 or 94. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 90% or at least 95% identical to the DNA- targeting segment set forth in SEQ ID NO: 62 or 94. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that differs by no more than 3, no more than 2, or no more than 1 nucleotide from the sequence set forth in SEQ ID NO: 62 or 94. [00302] As another example, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 65 or 97. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the DNA-targeting segment set forth in SEQ ID NO: 65 or 97. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 90% or at least 95% identical to the DNA- targeting segment set forth in SEQ ID NO: 65 or 97. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that differs by no more than 3, no more than 2, or no more than 1 nucleotide from the sequence set forth in SEQ ID NO: 65 or 97. [00303] As another example, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 73 or 105. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the DNA-targeting segment set forth in SEQ ID NO: 73 or 105. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that is at least 90% or at least 95% identical to the DNA- targeting segment set forth in SEQ ID NO: 73 or 105. Alternatively, a guide RNA targeting intron 1 of a human ALB gene can comprise, consist essentially of, or consist of a sequence that differs by no more than 3, no more than 2, or no more than 1 nucleotide from the sequence set forth in SEQ ID NO: 73 or 105. (4) Guide RNA Target Sequences [00304] Target DNAs for guide RNAs include nucleic acid sequences present in a DNA to which a DNA-targeting segment of a gRNA will bind, provided sufficient conditions for binding exist. Suitable DNA/RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA/RNA binding conditions (e.g., conditions in a cell-free system) are known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), herein incorporated by reference in its entirety for all purposes). The strand of the target DNA that is complementary to and hybridizes with the gRNA can be called the “complementary strand,” and the strand of the target DNA that is complementary to the “complementary strand” (and is therefore not complementary to the Cas protein or gRNA) can be called “noncomplementary strand” or “template strand.” [00305] The target DNA includes both the sequence on the complementary strand to which the guide RNA hybridizes and the corresponding sequence on the non-complementary strand (e.g., adjacent to the protospacer adjacent motif (PAM)). The term “guide RNA target sequence” as used herein refers specifically to the sequence on the non-complementary strand corresponding to (i.e., the reverse complement of) the sequence to which the guide RNA hybridizes on the complementary strand. That is, the guide RNA target sequence refers to the sequence on the non-complementary strand adjacent to the PAM (e.g., upstream or 5’ of the PAM in the case of Cas9). A guide RNA target sequence is equivalent to the DNA-targeting segment of a guide RNA, but with thymines instead of uracils. As one example, a guide RNA target sequence for an SpCas9 enzyme can refer to the sequence upstream of the 5’-NGG-3’ PAM on the non-complementary strand. A guide RNA is designed to have complementarity to the complementary strand of a target DNA, where hybridization between the DNA-targeting segment of the guide RNA and the complementary strand of the target DNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. If a guide RNA is referred to herein as targeting a guide RNA target sequence, what is meant is that the guide RNA hybridizes to the complementary strand sequence of the target DNA that is the reverse complement of the guide RNA target sequence on the non-complementary strand. [00306] A target DNA or guide RNA target sequence can comprise any polynucleotide, and can be located, for example, in the nucleus or cytoplasm of a cell or within an organelle of a cell, such as a mitochondrion or chloroplast. A target DNA or guide RNA target sequence can be any nucleic acid sequence endogenous or exogenous to a cell. The guide RNA target sequence can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory sequence) or can include both. [00307] Site-specific binding and cleavage of a target DNA by a Cas protein can occur at locations determined by both (i) base-pairing complementarity between the guide RNA and the complementary strand of the target DNA and (ii) a short motif, called the protospacer adjacent motif (PAM), in the non-complementary strand of the target DNA. The PAM can flank the guide RNA target sequence. Optionally, the guide RNA target sequence can be flanked on the 3’ end by the PAM (e.g., for Cas9). Alternatively, the guide RNA target sequence can be flanked on the 5’ end by the PAM (e.g., for Cpf1). For example, the cleavage site of Cas proteins can be about 1 to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence (e.g., within the guide RNA target sequence). In the case of SpCas9, the PAM sequence (i.e., on the non-complementary strand) can be 5’-N1GG-3’, where N1 is any DNA nucleotide, and where the PAM is immediately 3’ of the guide RNA target sequence on the non- complementary strand of the target DNA. As such, the sequence corresponding to the PAM on the complementary strand (i.e., the reverse complement) would be 5’-CCN 2 -3’, where N 2 is any DNA nucleotide and is immediately 5’ of the sequence to which the DNA-targeting segment of the guide RNA hybridizes on the complementary strand of the target DNA. In some such cases, N 1 and N 2 can be complementary and the N 1 - N 2 base pair can be any base pair (e.g., N 1 =C and N2=G; N1=G and N2=C; N1=A and N2=T; or N1=T, and N2=A). In the case of Cas9 from S. aureus, the PAM can be NNGRRT or NNGRR, where N can A, G, C, or T, and R can be G or A. In the case of Cas9 from C. jejuni, the PAM can be, for example, NNNNACAC or NNNNRYAC, where N can be A, G, C, or T, and R can be G or A. In some cases (e.g., for FnCpf1), the PAM sequence can be upstream of the 5’ end and have the sequence 5’-TTN-3’. In the case of DpbCasX, the PAM can have the sequence 5’-TTCN-3’. In the case of Cas^, the PAM can have the sequence 5’-TBN-3’, wherein B is G, T, or C. [00308] An example of a guide RNA target sequence is a 20-nucleotide DNA sequence immediately preceding an NGG motif recognized by an SpCas9 protein. For example, two examples of guide RNA target sequences plus PAMs are GN19NGG (SEQ ID NO: 5) or N20NGG (SEQ ID NO: 6). See, e.g., WO 2014/165825, herein incorporated by reference in its entirety for all purposes. The guanine at the 5’ end can facilitate transcription by RNA polymerase in cells. Other examples of guide RNA target sequences plus PAMs can include two guanine nucleotides at the 5’ end (e.g., GGN 20 NGG; SEQ ID NO: 7) to facilitate efficient transcription by T7 polymerase in vitro. See, e.g., WO 2014/065596, herein incorporated by reference in its entirety for all purposes. Other guide RNA target sequences plus PAMs can have between 4-22 nucleotides in length of SEQ ID NOS: 5-7, including the 5’ G or GG and the 3’ GG or NGG. Yet other guide RNA target sequences plus PAMs can have between 14 and 20 nucleotides in length of SEQ ID NOS: 5-7. [00309] Formation of a CRISPR complex hybridized to a target DNA can result in cleavage of one or both strands of the target DNA within or near the region corresponding to the guide RNA target sequence (i.e., the guide RNA target sequence on the non-complementary strand of the target DNA and the reverse complement on the complementary strand to which the guide RNA hybridizes). For example, the cleavage site can be within the guide RNA target sequence (e.g., at a defined location relative to the PAM sequence). The “cleavage site” includes the position of a target DNA at which a Cas protein produces a single-strand break or a double-strand break. The cleavage site can be on only one strand (e.g., when a nickase is used) or on both strands of a double-stranded DNA. Cleavage sites can be at the same position on both strands (producing blunt ends; e.g. Cas9)) or can be at different sites on each strand (producing staggered ends (i.e., overhangs); e.g., Cpf1). Staggered ends can be produced, for example, by using two Cas proteins, each of which produces a single-strand break at a different cleavage site on a different strand, thereby producing a double-strand break. For example, a first nickase can create a single- strand break on the first strand of double-stranded DNA (dsDNA), and a second nickase can create a single-strand break on the second strand of dsDNA such that overhanging sequences are created. In some cases, the guide RNA target sequence or cleavage site of the nickase on the first strand is separated from the guide RNA target sequence or cleavage site of the nickase on the second strand by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, or 1,000 base pairs. [00310] The guide RNA target sequence can also be selected to minimize off-target modification or avoid off-target effects (e.g., by avoiding two or fewer mismatches to off-target genomic sequences). [00311] As one example, a guide RNA targeting intron 1 of a human ALB gene can target the guide RNA target sequence set forth in any one of SEQ ID NOS: 126-157. As another example, a guide RNA targeting intron 1 of a human ALB gene can target at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the guide RNA target sequence set forth in any one of SEQ ID NOS: 126-157. [00312] As another example, a guide RNA targeting intron 1 of a human ALB gene can target the guide RNA target sequence set forth in any one of SEQ ID NOS: 132, 126, 129, and 137. As another example, a guide RNA targeting intron 1 of a human ALB gene can target at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the guide RNA target sequence set forth in any one of SEQ ID NOS: 132, 126, 129, and 137. [00313] As another example, a guide RNA targeting intron 1 of a human ALB gene can target the guide RNA target sequence set forth in SEQ ID NO: 132. As another example, a guide RNA targeting intron 1 of a human ALB gene can target at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the guide RNA target sequence set forth in SEQ ID NO: 132. [00314] As another example, a guide RNA targeting intron 1 of a human ALB gene can target the guide RNA target sequence set forth in SEQ ID NO: 126. As another example, a guide RNA targeting intron 1 of a human ALB gene can target at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the guide RNA target sequence set forth in SEQ ID NO: 126. [00315] As another example, a guide RNA targeting intron 1 of a human ALB gene can target the guide RNA target sequence set forth in SEQ ID NO: 129. As another example, a guide RNA targeting intron 1 of a human ALB gene can target at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the guide RNA target sequence set forth in SEQ ID NO: 129. [00316] As another example, a guide RNA targeting intron 1 of a human ALB gene can target the guide RNA target sequence set forth in SEQ ID NO: 137. As another example, a guide RNA targeting intron 1 of a human ALB gene can target at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the guide RNA target sequence set forth in SEQ ID NO: 137. [00317] Table 6. Human ALB Intron 1 Guide RNA Target Sequences. [00318] Table 7. Mouse Alb Intron 1 Guide RNA Target Sequences. (5) Lipid Nanoparticles Comprising Nuclease Agents [00319] Lipid nanoparticles comprising the nuclease agents (e.g., CRISPR/Cas systems) are also provided. The lipid nanoparticles can alternatively or additionally comprise a nucleic acid construct encoding a polypeptide of interest as disclosed herein. For example, the lipid nanoparticles can comprise a nuclease agent (e.g., CRISPR/Cas system), can comprise a nucleic acid construct encoding a polypeptide of interest, or can comprise both a nuclease agent (e.g., a CRISPR/Cas system) and a nucleic acid construct encoding a polypeptide of interest. Regarding CRISPR/Cas systems, the lipid nanoparticles can comprise the Cas protein in any form (e.g., protein, DNA, or mRNA) and/or can comprise the guide RNA(s) in any form (e.g., DNA or RNA). In one example, the lipid nanoparticles comprise the Cas protein in the form of mRNA (e.g., a modified RNA as described herein) and the guide RNA(s) in the form of RNA (e.g., a modified guide RNA as disclosed herein). As another example, the lipid nanoparticles can comprise the Cas protein in the form of protein and the guide RNA(s) in the form of RNA). In a specific example, the guide RNA and the Cas protein are each introduced in the form of RNA via LNP-mediated delivery in the same LNP. As discussed in more detail elsewhere herein, one or more of the RNAs can be modified. For example, guide RNAs can be modified to comprise one or more stabilizing end modifications at the 5’ end and/or the 3’ end. Such modifications can include, for example, one or more phosphorothioate linkages at the 5’ end and/or the 3’ end and/or one or more 2’-O-methyl modifications at the 5’ end and/or the 3’ end. As another example, Cas mRNA modifications can include substitution with pseudouridine (e.g., fully substituted with pseudouridine), 5’ caps, and polyadenylation. As another example, Cas mRNA modifications can include substitution with N1-methyl-pseudouridine (e.g., fully substituted with N1-methyl-pseudouridine), 5’ caps, and polyadenylation. Other modifications are also contemplated as disclosed elsewhere herein. Delivery through such methods can result in transient Cas expression and/or transient presence of the guide RNA, and the biodegradable lipids improve clearance, improve tolerability, and decrease immunogenicity. Lipid formulations can protect biological molecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles comprising a plurality of lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations which contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time for which nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO 2016/010840 A1 and WO 2017/173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. An exemplary lipid nanoparticle can comprise a cationic lipid and one or more other components. In one example, the other component can comprise a helper lipid such as cholesterol. In another example, the other components can comprise a helper lipid such as cholesterol and a neutral lipid such as distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In another example, the other components can comprise a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033. [00320] The LNP may contain one or more or all of the following: (i) a lipid for encapsulation and for endosomal escape; (ii) a neutral lipid for stabilization; (iii) a helper lipid for stabilization; and (iv) a stealth lipid. See, e.g., Finn et al. (2018) Cell Rep.22(9):2227-2235 and WO 2017/173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. In certain LNPs, the cargo can include a guide RNA or a nucleic acid encoding a guide RNA. In certain LNPs, the cargo can include an mRNA encoding a Cas nuclease, such as Cas9, and a guide RNA or a nucleic acid encoding a guide RNA. In certain LNPs, the cargo can include a nucleic acid construct encoding a polypeptide of interest as described elsewhere herein. In certain LNPs, the cargo can include an mRNA encoding a Cas nuclease, such as Cas9, a guide RNA or a nucleic acid encoding a guide RNA, and a nucleic acid construct encoding a polypeptide of. In some LNPs, the lipid component comprises an amine lipid such as a biodegradable, ionizable lipid. In some instances, the lipid component comprises biodegradable, ionizable lipid, cholesterol, DSPC, and PEG-DMG. For example, Cas9 mRNA and gRNA can be delivered to cells and animals utilizing lipid formulations comprising ionizable lipid ((9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)pro poxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG. [00321] In some examples, the LNPs comprise cationic lipids. In some examples, the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)car bonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g., WO 2019/067992, WO 2017/173054, WO 2015/095340, and WO 2014/136086, each of which is herein incorporated by reference in its entirety for all purposes. In some examples, the LNPs comprise molar ratios of a cationic lipid amine to RNA phosphate (N:P) of about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. In some examples, the terms cationic and ionizable in the context of LNP lipids are interchangeable (e.g., wherein ionizable lipids are cationic depending on the pH). [00322] The lipid for encapsulation and endosomal escape can be a cationic lipid. The lipid can also be a biodegradable lipid, such as a biodegradable ionizable lipid. One example of a suitable lipid is Lipid A or LP01, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)car bonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep.22(9):2227-2235 and WO 2017/173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. Another example of a suitable lipid is Lipid B, which is ((5-((dimethylamino)methyl)- 1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), also called ((5- ((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8, 1-diyl)bis(decanoate). Another example of a suitable lipid is Lipid C, which is 2-((4-(((3- (dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane -1,3-diyl(9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate). Another example of a suitable lipid is Lipid D, which is 3-(((3- (dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecy l 3-octylundecanoate. Other suitable lipids include heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (also known as [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate or Dlin-MC3-DMA (MC3))). [00323] Some such lipids suitable for use in the LNPs described herein are biodegradable in vivo. [00324] Such lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipids may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood where pH is approximately 7.35, the lipids may not be protonated and thus bear no charge. In some embodiments, the lipids may be protonated at a pH of at least about 9, 9.5, or 10. The ability of such a lipid to bear a charge is related to its intrinsic pKa. For example, the lipid may, independently, have a pKa in the range of from about 5.8 to about 6.2. [00325] Neutral lipids function to stabilize and improve processing of the LNPs. Examples of suitable neutral lipids include a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5- heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl- 2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and combinations thereof. For example, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). [00326] Helper lipids include lipids that enhance transfection. The mechanism by which the helper lipid enhances transfection can include enhancing particle stability. In certain cases, the helper lipid can enhance membrane fusogenicity. Helper lipids include steroids, sterols, and alkyl resorcinols. Examples of suitable helper lipids suitable include cholesterol, 5- heptadecylresorcinol, and cholesterol hemisuccinate. In one example, the helper lipid may be cholesterol or cholesterol hemisuccinate. [00327] Stealth lipids include lipids that alter the length of time the nanoparticles can exist in vivo. Stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids may modulate pharmacokinetic properties of the LNP. Suitable stealth lipids include lipids having a hydrophilic head group linked to a lipid moiety. [00328] The hydrophilic head group of stealth lipid can comprise, for example, a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids, and poly N-(2-hydroxypropyl)methacrylamide. The term PEG means any polyethylene glycol or other polyalkylene ether polymer. In certain LNP formulations, the PEG, is a PEG-2K, also termed PEG 2000, which has an average molecular weight of about 2,000 daltons. See, e.g., WO 2017/173054 A1, herein incorporated by reference in its entirety for all purposes. [00329] The lipid moiety of the stealth lipid may be derived, for example, from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. [00330] As one example, the stealth lipid may be selected from PEG-dilauroylglycerol, PEG- dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG- DSPE), PEG-dilaurylglycamide, PEG- dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'- dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4- ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn- glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k- DMPE),or 1,2- dimyristoyl-rac-glycero-3-methylpolyoxyethylene glycol-2000 (PEG2k-DMG), 1,2- distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypoly ethylene glycol (PEG2k-DSG), poly(ethylene glycol)- 2000-dimethacrylate (PEG2k-DMA), and 1,2- distearyloxypropyl-3-amine-N- [methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one particular example, the stealth lipid may be PEG2k-DMG. [00331] In some embodiments, the PEG lipid includes a glycerol group. In some embodiments, the PEG lipid includes a dimyristoylglycerol (DMG) group. In some embodiments, the PEG lipid comprises PEG2k. In some embodiments, the PEG lipid is a PEG- DMG. In some embodiments, the PEG lipid is a PEG2k-DMG. In some embodiments, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In some embodiments, the PEG2k-DMG is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. [00332] The LNPs can comprise different respective molar ratios of the component lipids in the formulation. The mol-% of the CCD lipid may be, for example, from about 30 mol-% to about 60 mol-%. The mol-% of the helper lipid may be, for example, from about 30 mol-% to about 60 mol-%. The mol-% of the neutral lipid may be, for example, from about 1 mol-% to about 20 mol-%. The mol-% of the stealth lipid may be, for example, from about 1 mol-% to about 10 mol-% [00333] The LNPs can have different ratios between the positively charged amine groups of the biodegradable lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N/P. For example, the N/P ratio may be from about 0.5 to about 100. The N/P ratio can also be from about 4 to about 6. [00334] In some LNPs, the cargo can comprise Cas mRNA (e.g., Cas9 mRNA) and gRNA. The Cas mRNA and gRNAs can be in different ratios. For example, the LNP formulation can include a ratio of Cas mRNA to gRNA nucleic acid ranging from about 25:1 to about 1:25. Alternatively, the LNP formulation can include a ratio of Cas mRNA to gRNA nucleic acid of from about 2:1 to about 1:2. In specific examples, the ratio of Cas mRNA to gRNA can be about 2:1. [00335] In some LNPs, the cargo can comprise a nucleic acid construct encoding a polypeptide of interest and gRNA. The nucleic acid construct encoding a polypeptide of interest and gRNAs can be in different ratios. For example, the LNP formulation can include a ratio of nucleic acid construct to gRNA nucleic acid ranging from about 25:1 to about 1:25. [00336] A specific example of a suitable LNP has a nitrogen-to-phosphate (N/P) ratio of about 4.5 and contains biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in an about 45:44:9:2 molar ratio (about 45:about 44:about 9:about 2). The biodegradable cationic lipid can be (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)car bonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep.22(9):2227-2235, herein incorporated by reference in its entirety for all purposes. The Cas9 mRNA can be in an about 1:1 (about 1:about 1) ratio by weight to the guide RNA. Another specific example of a suitable LNP contains Dlin-MC3-DMA (MC3), cholesterol, DSPC, and PEG-DMG in an about 50:38.5:10:1.5 molar ratio (about 50:about 38.5:about 10:about 1.5). The Cas9 mRNA can be in an about 1:2 ratio (about 1:about 2)by weight to the guide RNA. The Cas9 mRNA can be in an about 1:1 ratio (about 1:about 1) by weight to the guide RNA. The Cas9 mRNA can be in an about 2:1 ratio (about 2:about 1) by weight to the guide RNA. [00337] Another specific example of a suitable LNP has a nitrogen-to-phosphate (N/P) ratio of about 6 and contains biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in an about 50:38:9:3 molar ratio (about 50:about 38:about 9:about 3). The biodegradable cationic lipid can be Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)car bonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate). The Cas9 mRNA can be in an about 1:2 ratio (about 1:about 2) by weight to the guide RNA. The Cas9 mRNA can be in an about 1:1 ratio (about 1:about 1)by weight to the guide RNA. The Cas9 mRNA can be in an about 2:1 (about 2:about 1) ratio by weight to the guide RNA. [00338] Another specific example of a suitable LNP has a nitrogen-to-phosphate (N/P) ratio of about 3 and contains a cationic lipid, a structural lipid, cholesterol (e.g., cholesterol (ovine) (Avanti 700000)), and PEG2k-DMG (e.g., PEG-DMG 2000 (NOF America-SUNBRIGHT ® GM-020(DMG-PEG)) in an about 50:10:38.5:1.5 ratio (about 50:about 10:about 38.5:about 1.5) or an about 47:10:42:1 ratio (about 47:about 10:about 42:about 1). The structural lipid can be, for example, DSPC (e.g., DSPC (Avanti 850365)), SOPC, DOPC, or DOPE. The cationic/ionizable lipid can be, for example, Dlin-MC3-DMA (e.g., Dlin-MC3-DMA (Biofine International)). The Cas9 mRNA can be in an about 1:2 ratio (about 1:about 2) by weight to the guide RNA. The Cas9 mRNA can be in an about 1:1 ratio (about 1:about 1) by weight to the guide RNA. The Cas9 mRNA can be in an about 2:1 ratio (about 2:about 1) by weight to the guide RNA. [00339] Another specific example of a suitable LNP contains Dlin-MC3-DMA, DSPC, cholesterol, and a PEG lipid in an about 45:9:44:2 ratio (about 45:about 9:about 44:about 2). Another specific example of a suitable LNP contains Dlin-MC3-DMA, DOPE, cholesterol, and PEG lipid or PEG DMG in an about 50:10:39:1 ratio (about 50:about 10:about 39:about 1). Another specific example of a suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol, and PEG2k-DMG at an about 55:10:32.5:2.5 ratio (about 55:about 10:about 32.5:about 2.5). Another specific example of a suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in an about 50:10:38.5:1.5 ratio (about 50:about 10:about 38.5:about 1.5). Another specific example of a suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in an about 50:10:38.5:1.5 ratio (about 50:about 10:about 38.5:about 1.5). The Cas9 mRNA can be in an about 1:2 ratio (about 1:about 2) by weight to the guide RNA. The Cas9 mRNA can be in an about 1:1 ratio (about 1:about 1) by weight to the guide RNA. The Cas9 mRNA can be in an about 2:1 ratio (about 2:about 1) by weight to the guide RNA. [00340] Other examples of suitable LNPs can be found, e.g., in WO 2019/067992, WO 2020/082042, US 2020/0270617, WO 2020/082041, US 2020/0268906, WO 2020/082046 (see, e.g., pp.85-86), and US 2020/0289628, each of which is herein incorporated by reference in its entirety for all purposes. (6) Vectors Comprising Nuclease Agents [00341] The nuclease agents disclosed herein (e.g., ZFN, TALEN, or CRISPR/Cas) can be provided in a vector for expression. A vector can comprise additional sequences such as, for example, replication origins, promoters, and genes encoding antibiotic resistance. [00342] Some vectors may be circular. Alternatively, the vector may be linear. The vector can be in the packaged for delivered via a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors. [00343] Introduction of nucleic acids can also be accomplished by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. The vectors can be, for example, viral vectors such as adeno-associated virus (AAV) vectors. The AAV may be any suitable serotype and may be a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV). Other exemplary viruses/viral vectors include retroviruses, lentiviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The viruses can integrate into the host genome or alternatively do not integrate into the host genome. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or can be replication-defective (e.g., defective in one or more genes necessary for additional rounds of virion replication and/or packaging). Viral vector may be genetically modified from their wild type counterparts. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some examples, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some examples, the viral vector may have an enhanced transduction efficiency. In some examples, the immune response induced by the virus in a host may be reduced. In some examples, viral genes (such as integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some examples, the viral vector may be replication defective. In some examples, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some examples, the virus may be helper-dependent. For example, the virus may need one or more helper virus to supply viral components (such as viral proteins) required to amplify and package the vectors into viral particles. In such a case, one or more helper components, including one or more vectors encoding the viral components, may be introduced into a host cell or population of host cells along with the vector system described herein. In other examples, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without a helper virus. In some examples, the vector system described herein may also encode the viral components required for virus amplification and packaging. [00344] Exemplary viral titers (e.g., AAV titers) include about 10 12 to about 10 16 vg/mL. Other exemplary viral titers (e.g., AAV titers) include about 10 12 to about 10 16 vg/kg of body weight. [00345] Adeno-associated viruses (AAVs) are endemic in multiple species including human and non-human primates (NHPs). At least 12 natural serotypes and hundreds of natural variants have been isolated and characterized to date. See, e.g., Li et al. (2020) Nat. Rev. Genet.21:255- 272, herein incorporated by reference in its entirety for all purposes. AAV particles are naturally composed of a non-enveloped icosahedral protein capsid containing a single-stranded DNA (ssDNA) genome. The DNA genome is flanked by two inverted terminal repeats (ITRs) which serve as the viral origins of replication and packaging signals. The rep gene encodes four proteins required for viral replication and packaging whilst the cap gene encodes the three structural capsid subunits which dictate the AAV serotype, and the Assembly Activating Protein (AAP) which promotes virion assembly in some serotypes. [00346] Recombinant AAV (rAAV) is currently one of the most commonly used viral vectors used in gene therapy to treat human diseases by delivering therapeutic transgenes to target cells in vivo. Indeed, rAAV vectors are composed of icosahedral capsids similar to natural AAVs, but rAAV virions do not encapsidate AAV protein-coding or AAV replicating sequences. These viral vectors are non-replicating. The only viral sequences required in rAAV vectors are the two ITRs, which are needed to guide genome replication and packaging during manufacturing of the rAAV vector. rAAV genomes are devoid of AAV rep and cap genes, rendering them non- replicating in vivo. rAAV vectors are produced by expressing rep and cap genes along with additional viral helper proteins in trans, in combination with the intended transgene cassette flanked by AAV ITRs. [00347] In therapeutic rAAV genomes, a gene expression cassette is placed between ITR sequences. Typically, rAAV genome cassettes comprise of a promoter to drive expression of a therapeutic transgene, followed by polyadenylation sequence. The ITRs flanking a rAAV expression cassette are usually derived from AAV2, the first serotype to be isolated and converted into a recombinant viral vector. Since then, most rAAV production methods rely on AAV2 Rep-based packaging systems. See, e.g., Colella et al. (2017) Mol. Ther. Methods Clin. Dev.8:87-104, herein incorporated by reference in its entirety for all purposes. [00348] Some non-limiting examples of ITRs that can be used include ITRs comprising, consisting essentially of, or consisting of SEQ ID NO: 158, SEQ ID NO: 159, or SEQ ID NO: 160. Other examples of ITRs comprise one or more mutations compared to SEQ ID NO: 158, SEQ ID NO: 159, or SEQ ID NO: 160 and can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 158, SEQ ID NO: 159, or SEQ ID NO: 160. In some rAAV genomes disclosed herein, the nucleic acid encoding the nuclease agent (or component thereof) is flanked on both sides by the same ITR (i.e., the ITR on the 5’ end, and the reverse complement of the ITR on the 3’ end, such as SEQ ID NO: 158 on the 5’ end and SEQ ID NO: 168 on the 3’ end, or SEQ ID NO: 159 on the 5’ end and SEQ ID NO: 597 on the 3’ end, or SEQ ID NO: 160 on the 5’ end and SEQ ID NO: 598 on the 3’ end). In one example, the ITR on each end can comprise, consist essentially of, or consist of SEQ ID NO: 158 (i.e., SEQ ID NO: 158 on the 5’ end, and the reverse complement on the 3’ end). In another example, the ITR on each end can comprise, consist essentially of, or consist of SEQ ID NO: 159 (i.e., SEQ ID NO: 159 on the 5’ end, and the reverse complement on the 3’ end). In one example, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 160. In one example, the ITR on the 5’ end comprises, consists essentially of, or consists of SEQ ID NO: 160. In one example, the ITR on the 3’ end comprises, consists essentially of, or consists of SEQ ID NO: 160. In one example, the ITR on each end can comprise, consist essentially of, or consist of SEQ ID NO: 160 (i.e., SEQ ID NO: 160 on the 5’ end, and the reverse complement on the 3’ end). In one example, the ITR on each end can comprise, consist essentially of, or consist of SEQ ID NO: 160. In other rAAV genomes disclosed herein, the nucleic acid encoding the nuclease agent (or component thereof) is flanked by different ITRs on each end. In one example, the ITR on one end comprises, consists essentially of, or consists of SEQ ID NO: 158, and the ITR on the other end comprises, consists essentially of, or consists of SEQ ID NO: 159. In another example, the ITR on one end comprises, consists essentially of, or consists of SEQ ID NO: 158, and the ITR on the other end comprises, consists essentially of, or consists of SEQ ID NO: 160. In one example, the ITR on one end comprises, consists essentially of, or consists of SEQ ID NO: 159, and the ITR on the other end comprises, consists essentially of, or consists of SEQ ID NO: 160. [00349] The specific serotype of a recombinant AAV vector influences its in vivo tropism to specific tissues. AAV capsid proteins are responsible for mediating attachment and entry into target cells, followed by endosomal escape and trafficking to the nucleus. Thus, the choice of serotype when developing a rAAV vector will influence what cell types and tissues the vector is most likely to bind to and transduce when injected in vivo. Several serotypes of rAAVs, including rAAV8, are capable of transducing the liver when delivered systemically in mice, NHPs and humans. See, e.g., Li et al. (2020) Nat. Rev. Genet.21:255-272, herein incorporated by reference in its entirety for all purposes. [00350] Once in the nucleus, the ssDNA genome is released from the virion and a complementary DNA strand is synthesized to generate a double-stranded DNA (dsDNA) molecule. Double-stranded AAV genomes naturally circularize via their ITRs and become episomes which will persist extrachromosomally in the nucleus. Therefore, for episomal gene therapy programs, rAAV-delivered rAAV episomes provide long-term, promoter-driven gene expression in non-dividing cells. However, this rAAV-delivered episomal DNA is diluted out as cells divide. In contrast, the gene therapy described herein is based on gene insertion to allow long-term gene expression. [00351] When specific rAAVs comprising specific sequences (e.g., specific bidirectional construct sequences or specific unidirectional construct sequences) are disclosed herein, they are meant to encompass the sequence disclosed or the reverse complement of the sequence. For example, if a bidirectional or unidirectional construct disclosed herein consists of the hypothetical sequence 5’-CTGGACCGA-3’, it is also meant to encompass the reverse complement of that sequence (5’-TCGGTCCAG-3’). Likewise, when rAAVs comprising bidirectional or unidirectional construct elements in a specific 5’ to 3’ order are disclosed herein, they are also meant to encompass the reverse complement of the order of those elements. For example, if an rAAV is disclosed herein that comprises a bidirectional construct that comprises from 5’ to 3’ a first splice acceptor, a first coding sequence, a first terminator, a reverse complement of a second terminator, a reverse complement of a second coding sequence, and a reverse complement of a second splice acceptor, it is also meant to encompass a construct comprising from 5’ to 3’ the second splice acceptor, the second coding sequence, the second terminator, a reverse complement of the first terminator, a reverse complement of the first coding sequence, and a reverse complement of the first splice acceptor. Single-stranded AAV genomes are packaged as either sense (plus-stranded) or anti-sense (minus-stranded genomes), and single- stranded AAV genomes of + and – polarity are packaged with equal frequency into mature rAAV virions. See, e.g., LING et al. (2015) J. Mol. Genet. Med.9(3):175, Zhou et al. (2008) Mol. Ther.16(3):494-499, and Samulski et al. (1987) J. Virol.61:3096-3101, each of which is herein incorporated by reference in its entirety for all purposes. [00352] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats that allow for synthesis of the complementary DNA strand. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be supplied in trans. In addition to Rep and Cap, AAV can require a helper plasmid containing genes from adenovirus. These genes (E4, E2a, and VA) mediate AAV replication. For example, the transfer plasmid, Rep/Cap, and the helper plasmid can be transfected into HEK293 cells containing the adenovirus gene E1+ to produce infectious AAV particles. Alternatively, the Rep, Cap, and adenovirus helper genes may be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses. [00353] Multiple serotypes of AAV have been identified. These serotypes differ in the types of cells they infect (i.e., their tropism), allowing preferential transduction of specific cell types. The term AAV includes, for example, AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. A “AAV vector” as used herein refers to an AAV vector comprising a heterologous sequence not of AAV origin (i.e., a nucleic acid sequence heterologous to AAV), typically comprising a sequence encoding an exogenous polypeptide of interest. The construct may comprise an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV capsid sequence. In general, the heterologous nucleic acid sequence (the transgene) is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs). An AAV vector may either be single-stranded (ssAAV) or self-complementary (scAAV). Examples of serotypes for liver tissue include AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.74, and AAVhu.37, and particularly AAV8. In a specific example, the AAV vector comprising the nucleic acid construct can be recombinant AAV8 (rAAV8). A rAAV8 vector as described herein is one in which the capsid is from AAV8. For example, an AAV vector using ITRs from AAV2 and a capsid of AAV8 is considered herein to be a rAAV8 vector. [00354] Tropism can be further refined through pseudotyping, which is the mixing of a capsid and a genome from different viral serotypes. For example AAV2/5 indicates a virus containing the genome of serotype 2 packaged in the capsid from serotype 5. Use of pseudotyped viruses can improve transduction efficiency, as well as alter tropism. Hybrid capsids derived from different serotypes can also be used to alter viral tropism. For example, AAV-DJ contains a hybrid capsid from eight serotypes and displays high infectivity across a broad range of cell types in vivo. AAV-DJ8 is another example that displays the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified through mutations. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V. Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of mutational modifications of AAV6 include S663V and T492V. Other pseudotyped/modified AAV variants include AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5, AAV8.2, and AAV/SASTG. [00355] To accelerate transgene expression, self-complementary AAV (scAAV) variants can be used. Because AAV depends on the cell’s DNA replication machinery to synthesize the complementary strand of the AAV’s single-stranded DNA genome, transgene expression may be delayed. To address this delay, scAAV containing complementary sequences that are capable of spontaneously annealing upon infection can be used, eliminating the requirement for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used. [00356] To increase packaging capacity, longer transgenes may be split between two AAV transfer plasmids, the first with a 3’ splice donor and the second with a 5’ splice acceptor. Upon co-infection of a cell, these viruses form concatemers, are spliced together, and the full-length transgene can be expressed. Although this allows for longer transgene expression, expression is less efficient. Similar methods for increasing capacity utilize homologous recombination. For example, a transgene can be divided between two transfer plasmids but with substantial sequence overlap such that co-expression induces homologous recombination and expression of the full- length transgene. [00357] In certain AAVs, the cargo can include nucleic acids encoding one or more guide RNAs (e.g., DNA encoding a guide RNA, or DNA encoding two or more guide RNAs). In certain AAVs, the cargo can include a nucleic acid (e.g., DNA) encoding a Cas nuclease, such as Cas9, and DNA encoding one or more guide RNAs (e.g., DNA encoding a guide RNA, or DNA encoding two or more guide RNAs). In certain AAVs, the cargo can include a nucleic acid construct encoding a polypeptide of interest. In certain AAVs, the cargo can include a nucleic acid (e.g., DNA) encoding a Cas nuclease, such as Cas9, a DNA encoding a guide RNA (or multiple guide RNAs), and a nucleic acid construct encoding a polypeptide of interest. [00358] For example, Cas or Cas9 and one or more gRNAs (e.g., 1 gRNA or 2 gRNAs or 3 gRNAs or 4 gRNAs) can be delivered via LNP-mediated delivery (e.g., in the form of RNA) or adeno-associated virus (AAV)-mediated delivery (e.g., rAAV8-mediated delivery). For example, a Cas9 mRNA and a gRNA can be delivered via LNP-mediated delivery, or DNA encoding Cas9 and DNA encoding a gRNA can be delivered via AAV-mediated delivery. The Cas or Cas9 and the gRNA(s) can be delivered in a single AAV or via two separate AAVs. For example, a first AAV can carry a Cas or Cas9 expression cassette, and a second AAV can carry a gRNA expression cassette. Similarly, a first AAV can carry a Cas or Cas9 expression cassette, and a second AAV can carry two or more gRNA expression cassettes. Alternatively, a single AAV can carry a Cas or Cas9 expression cassette (e.g., Cas or Cas9 coding sequence operably linked to a promoter) and a gRNA expression cassette (e.g., gRNA coding sequence operably linked to a promoter). Similarly, a single AAV can carry a Cas or Cas9 expression cassette (e.g., Cas or Cas9 coding sequence operably linked to a promoter) and two or more gRNA expression cassettes (e.g., gRNA coding sequences operably linked to promoters). Different promoters can be used to drive expression of the gRNA, such as a U6 promoter or the small tRNA Gln. Likewise, different promoters can be used to drive Cas9 expression. For example, small promoters are used so that the Cas9 coding sequence can fit into an AAV construct. Similarly, small Cas9 proteins (e.g., SaCas9 or CjCas9 are used to maximize the AAV packaging capacity). C. Cells or Animals or Genomes [00359] Cells or animals (i.e., subjects) comprising any of the above compositions (e.g., nucleic acid construct encoding a polypeptide of interest, nuclease agents, vectors, lipid nanoparticles, or any combination thereof) are also provided herein. Such cells or animals (or genomes) can be produced by the methods disclosed herein. For example, the cells or animals can comprise any of the nucleic acid constructs encoding a polypeptide of interest described herein, any of the nuclease agents disclosed herein, or both. Such cells or animals (or genomes) can be neonatal cells or animals (or genomes). Alternatively, such cells or animals (or genomes) can be non-neonatal cells or animals (or genomes). [00360] A neonatal subject (e.g., animal) can be a human subject up to or under the age of 1 year (52 weeks), preferably up to or under the age of 24 weeks, more preferably up to or under the age of 12 weeks, more preferably up to or under the age of 8 weeks, and even more preferably up to or under the age of 4 weeks. In certain embodiments, a neonatal human subject is up to 4 weeks of age. In certain embodiments, a neonatal human subject is up to 8 weeks of age. In another embodiment, a neonatal human subject is within 3 weeks after birth. In another embodiment, a neonatal human subject is within 2 weeks after birth. In another embodiment, a neonatal human subject is within 1 week after birth. In another embodiment, a neonatal human subject is within 7 days after birth. In another embodiment, a neonatal human subject is within 6 days after birth. In another embodiment, a neonatal human subject is within 5 days after birth. In another embodiment, a neonatal human subject is within 4 days after birth. In another embodiment, a neonatal human subject is within 3 days after birth. In another embodiment, a neonatal human subject is within 2 days after birth. In another embodiment, a neonatal human subject is within 1 day after birth. The time windows disclosed above are for human subjects and are also meant to cover the corresponding developmental time windows for other animals. [00361] Neonatal cells can be cells of any neonatal subject. For example, they can be of a human subject up to or under the age of 1 year (52 weeks), preferably up to or under the age of 24 weeks, more preferably up to or under the age of 12 weeks, more preferably up to or under the age of 8 weeks, and even more preferably up to or under the age of 4 weeks. In certain embodiments, a neonatal human subject is up to 4 weeks of age. In certain embodiments, a neonatal human subject is up to 8 weeks of age. In another embodiment, a neonatal human subject is within 3 weeks after birth. In another embodiment, a neonatal human subject is within 2 weeks after birth. In another embodiment, a neonatal human subject is within 1 week after birth. In another embodiment, a neonatal human subject is within 7 days after birth. In another embodiment, a neonatal human subject is within 6 days after birth. In another embodiment, a neonatal human subject is within 5 days after birth. In another embodiment, a neonatal human subject is within 4 days after birth. In another embodiment, a neonatal human subject is within 3 days after birth. In another embodiment, a neonatal human subject is within 2 days after birth. In another embodiment, a neonatal human subject is within 1 day after birth. The time windows disclosed above are for human subjects and are also meant to cover the corresponding developmental time windows for other animals. [00362] In some such cells or animals or genomes, the nucleic acid construct encoding a polypeptide of interest can be genomically integrated at a target genomic locus, such as a safe harbor locus (e.g., an ALB locus or a human ALB locus, such as intron 1 of an ALB locus or a human ALB locus). In some such cells, animals, or genomes, the polypeptide of interest encoded by the nucleic acid construct is expressed in the cell, animal, or genome. For example, if the nucleic acid construct encoding a polypeptide of interest is integrated into an ALB locus (e.g., intron 1 of a human ALB locus), the polypeptide of interest can be expressed from the ALB locus. The coding sequence for the polypeptide of interest can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. If the nucleic acid construct is a bidirectional nucleic acid construct disclosed herein, the neonatal genome, neonatal cell, or neonatal animal can express the first polypeptide of interest or can express the second polypeptide of interest. In some neonatal genomes, neonatal cells, or neonatal animals, the target genomic locus is an ALB locus. For example, the nucleic acid construct can be genomically integrated in intron 1 of the endogenous ALB locus. Endogenous ALB exon 1 can then splice into the coding sequence for the polypeptide of interest in the nucleic acid construct. [00363] The target genomic locus at which the nucleic acid construct is stably integrated can be heterozygous for the nucleic acid construct encoding a polypeptide of interest or homozygous for the nucleic acid construct encoding a polypeptide of interest. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. [00364] The cells, neonatal, or genomes can be from any suitable species, such as eukaryotic cells or eukaryotes, or mammalian cells or mammals (e.g., non-human mammalian cells or non- human mammals, or human cells or humans). A mammal can be, for example, a non-human mammal, a human, a rodent, a rat, a mouse, or a hamster. Other non-human mammals include, for example, non-human primates, e.g., monkeys and apes. The term “non-human” excludes humans. Examples include, but are not limited to, human cells/humans, rodent cells/rodents, mouse cells/mice, rat cells/rats, and non-human primate cells/non-human primates. In a specific example, the cell is a human cell or the animal is a human. Likewise, cells can be any suitable type of cell. In a specific example, the cell is a liver cell such as a hepatocyte (e.g., a human liver cell or human hepatocyte). [00365] The cells can be isolated cells (e.g., in vitro), ex vivo cells, or can be in vivo within an animal (i.e., in a subject). The cells can be mitotically competent cells or mitotically-inactive cells, meiotically competent cells or meiotically-inactive cells. Similarly, the cells can also be primary somatic cells or cells that are not a primary somatic cell. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. For example, the neonatal cells can be liver cells, such as hepatocytes (e.g., mouse, non-human primate, or human hepatocytes). [00366] The cells provided herein can be normal, healthy cells, or can be diseased or mutant- bearing cells. For example, the cells can have a deficiency of the polypeptide of interest or can be from a subject with deficiency of the polypeptide of interest. For example, the cells can have a GAA deficiency, can carry a mutation that results in a GAA deficiency, or can be from a subject with a GAA deficiency carrying a mutation that results in a GAA deficiency, or Pompe disease. In some embodiments, the cells are of a neonatal subject. [00367] The cells provided herein can be dividing cells (e.g., actively dividing cells). Alternatively, the cells provided herein can be non-dividing cells. III. Therapeutic Methods and Methods for Introducing, Integrating, or Expressing a Nucleic Acid Encoding a Polypeptide of Interest in Cells or Subjects [00368] The nucleic acid constructs and compositions disclosed herein can be used in methods of inserting or integrating a nucleic acid encoding a polypeptide of interest into a target genomic locus or methods of expressing a polypeptide of interest in a cell, in a population of cells, or in a subject (e.g., in a neonatal cell, in a population of neonatal cells, or in a neonatal subject). [00369] The cells or populations of cells in the methods disclosed herein can be neonatal cells or populations of neonatal cells, and the subjects in the methods disclosed herein can be neonatal subjects in some methods. A neonatal subject can be a human subject up to or under the age of 1 year (52 weeks), preferably up to or under the age of 24 weeks, more preferably up to or under the age of 12 weeks, more preferably up to or under the age of 8 weeks, and even more preferably up to or under the age of 4 weeks. In certain embodiments, a neonatal human subject is up to 4 weeks of age. In certain embodiments, a neonatal human subject is up to 8 weeks of age. In another embodiment, a neonatal human subject is within 3 weeks after birth. In another embodiment, a neonatal human subject is within 2 weeks after birth. In another embodiment, a neonatal human subject is within 1 week after birth. In another embodiment, a neonatal human subject is within 7 days after birth. In another embodiment, a neonatal human subject is within 6 days after birth. In another embodiment, a neonatal human subject is within 5 days after birth. In another embodiment, a neonatal human subject is within 4 days after birth. In another embodiment, a neonatal human subject is within 3 days after birth. In another embodiment, a neonatal human subject is within 2 days after birth. In another embodiment, a neonatal human subject is within 1 day after birth. The time windows disclosed above are for human subjects and are also meant to cover the corresponding developmental time windows for other animals. As used herein, a “neonatal cell” is a cell of a neonatal subject, and a population of neonatal cells is a population of cells of a neonatal subject. In other methods, the cells or populations of cells are not neonatal cells and are not populations of neonatal cells, and the subjects are not neonatal subjects. [00370] In one example, provided herein are methods of introducing a nucleic acid construct encoding a polypeptide of interest into a cell or a population of cells, such as a cell or a population of cells in a subject (e.g., neonatal cell or a population of neonatal cells, such as a neonatal cell or a population of neonatal cells in a neonatal subject). Such methods can comprise administering any of the nucleic acid constructs described herein (or any of the compositions comprising a nucleic acid construct described herein, including, for example, vectors or lipid nanoparticles) to the cell, the population of cells, or the subject (e.g., the neonatal cell, the population of neonatal cells, or the neonatal subject). In some methods, the nucleic acid construct or composition comprising the nucleic acid construct can be administered together with a nuclease agent (simultaneously or sequentially in any order) described herein. The nuclease agent can cleave a nuclease target sequence within a target genomic locus (e.g., target gene) (e.g., to create a cleavage site), and the nucleic acid construct can be inserted into the target genomic locus (e.g., into the cleavage site) to create a modified target genomic locus. The polypeptide of interest can be expressed from the modified target genomic locus. The coding sequence for the polypeptide of interest can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. In one example, the nuclease agent is a CRISPR/Cas system, and the target gene is ALB (e.g., intron 1 of ALB). In such methods, the guide RNA can bind to the Cas protein and target the Cas protein to the guide RNA target sequence in intron 1 of the ALB gene, the Cas protein can cleave the guide RNA target sequence (e.g., to create a cleavage site), the nucleic acid construct can be inserted into ALB intron 1 (e.g., into the cleavage site) to create a modified ALB gene, and polypeptide of interest can be expressed from the modified ALB gene. [00371] In one example, provided herein are methods of inserting a nucleic acid construct encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells, such as a cell or a population of cells in a subject (e.g., in a neonatal cell or a population of neonatal cells, such as a neonatal cell or a population of neonatal cells in a neonatal subject). Such methods can comprise administering any of the nucleic acid constructs described herein (or any of the compositions comprising a nucleic acid construct described herein, including, for example, vectors or lipid nanoparticles) to the cell, the population of cells, or the subject (e.g., the neonatal cell, the population of neonatal cells, or the neonatal subject). In some methods, the nucleic acid construct or composition comprising the nucleic acid construct can be administered together with a nuclease agent (simultaneously or sequentially in any order) described herein. The nuclease agent can cleave a nuclease target sequence within a target genomic locus (e.g., target gene) (e.g., to create a cleavage site), and the nucleic acid construct can be inserted into the target genomic locus (e.g., into the cleavage site) to create a modified target genomic locus. The polypeptide of interest can be expressed from the modified target genomic locus. The coding sequence for the polypeptide of interest can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. In one example, the nuclease agent is a CRISPR/Cas system, and the target gene is ALB (e.g., intron 1 of ALB). In such methods, the guide RNA can bind to the Cas protein and target the Cas protein to the guide RNA target sequence in intron 1 of the ALB gene, the Cas protein can cleave the guide RNA target sequence (e.g., to create a cleavage site), the nucleic acid construct can be inserted into ALB intron 1 (e.g., into the cleavage site) to create a modified ALB gene, and polypeptide of interest can be expressed from the modified ALB gene. [00372] In another example, provided herein are methods of expressing a polypeptide of interest from a target genomic locus in a cell, a population of cells, or a subject (e.g., in a neonatal cell, a population of neonatal cells, or a neonatal subject). Such methods can comprise administering any of the nucleic acid constructs described herein (or any of the compositions comprising a nucleic acid construct described herein, including, for example, vectors or lipid nanoparticles) to the cell, the population of cells, or the subject (e.g., to the neonatal cell, the population of neonatal cells, or the neonatal subject). In some methods, the nucleic acid construct can be administered together (simultaneously or sequentially in any order) with a nuclease agent described herein. The nuclease agent can cleave a nuclease target sequence within a target genomic locus (e.g., target gene) (e.g., to create a cleavage site), the nucleic acid construct can be inserted into the target genomic locus (e.g., into the cleavage site) to create a modified target genomic locus, and the polypeptide of interest can be expressed from the modified target genomic locus. The coding sequence for the polypeptide of interest can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. In one example, the nuclease agent is a CRISPR/Cas system, and the target gene is ALB (e.g., intron 1 of ALB). In such methods, the guide RNA can bind to the Cas protein and target the Cas protein to the guide RNA target sequence in intron 1 of the ALB gene, the Cas protein can cleave the guide RNA target sequence (e.g., to create a cleavage site), the nucleic acid construct can be inserted into the target genomic locus (e.g., into the cleavage site) to create a modified ALB gene, and the polypeptide of interest can be expressed from the modified ALB gene. [00373] In some methods, the subject comprises a mutation in a genome in the subject, wherein the mutation results in reduced activity or expression of an endogenous polypeptide having enzymatic activity. In some methods, the nucleic acid encoding the polypeptide of interest encodes a polypeptide having the enzymatic activity of a wild type polypeptide encoded by the gene in which the subject has a mutation that results in reduced activity or expression of the endogenous polypeptide. [00374] In any of the above methods, the cells (e.g., neonatal cells) can be from any suitable species, such as eukaryotic cells or mammalian cells (e.g., non-human mammalian cells or human cells). A mammal can be, for example, a non-human mammal, a human, a rodent, a rat, a mouse, or a hamster. Other non-human mammals include, for example, non-human primates, e.g., monkeys and apes. The term “non-human” excludes humans. Specific examples of cells (e.g., neonatal cells) include, but are not limited to, human cells, rodent cells, mouse cells, rat cells, and non-human primate cells. In a specific example, the cell (e.g., neonatal cell) is a human cell. Likewise, cells (e.g., neonatal cells) can be any suitable type of cell. In a specific example, the cell (e.g., neonatal cell) is a liver cell such as a hepatocyte (e.g., a human liver cell or human hepatocyte). [00375] The cells (e.g., neonatal cells) can be isolated cells (e.g., in vitro), ex vivo cells, or can be in vivo within an animal (i.e., in a subject or a neonatal subject). In a specific example, the cell or neonatal cell is in vivo (in a subject or neonatal subject). Similarly, the cells or neonatal cells can also be primary somatic cells or cells that are not a primary somatic cell. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. For example, the neonatal cells can be liver cells, such as hepatocytes (e.g., mouse, non-human primate, or human hepatocytes). [00376] The cells (e.g., neonatal cells) provided herein can be normal, healthy cells, or can be diseased or mutant-bearing cells. In certain embodiments, the cells may demonstrate a loss of function, e.g., a loss of enzyme function. [00377] The nucleic acid constructs and compositions disclosed herein can also be used in methods of treating an enzyme deficiency and methods of treating a lysosomal storage disease in a subject (e.g., a neonatal subject). The nucleic acid constructs and compositions disclosed herein can also be used in methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency and or a lysosomal storage disease in a subject (e.g., a neonatal subject). [00378] The nucleic acid constructs and compositions disclosed herein can also be used in methods of treating a genetic disease that can be detected, including those that are routinely screened for, in newborn screening in a subject (e.g., a neonatal subject). The nucleic acid constructs and compositions disclosed herein can also be used in methods of preventing or reducing the onset of a sign or symptom of such diseases in a subject (e.g., a neonatal subject). [00379] The nucleic acid constructs and compositions disclosed herein can also be used in methods of treating inborn errors of metabolism in a subject (e.g., a neonatal subject). The nucleic acid constructs and compositions disclosed herein can also be used in methods of preventing or reducing the onset of a sign or symptom of diseases associated with inborn errors of metabolism in a subject (e.g., a neonatal subject). [00380] The nucleic acid constructs and compositions disclosed herein can also be used in methods of treating bleeding disorders in a subject (e.g., a neonatal subject). The nucleic acid constructs and compositions disclosed herein can also be used in methods of preventing or reducing the onset of a sign or symptom of bleeding disorders in a subject (e.g., a neonatal subject). [00381] The compositions disclosed herein (e.g., nucleic acid constructs encoding a polypeptide of interest, or nucleic acid constructs in combination with the nuclease agents (e.g., CRISPR/Cas systems) are useful for the treatment of enzyme deficiencies or lysosomal storage diseases and/or ameliorating at least one symptom associated with enzyme deficiencies or lysosomal storage diseases. Likewise, the compositions disclosed herein can be used for the preparation of a pharmaceutical composition or medicament for treating a subject (e.g., a neonatal subject) having an enzyme deficiency or lysosomal storage disease. The terms “treat,” “treated,” “treating,” and “treatment,” include the administration of the nucleic acid constructs disclosed herein (e.g., together with a nuclease agent disclosed herein) to subjects to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. It is understood that a number of lysosomal storage diseases or inborn diseases of metabolism are possible to diagnose before the presence of symptoms, or diagnosed through routine newborn screening programs, including pilot programs. Some include diagnosis based on the presence of a biomarker, e.g., a metabolite or enzyme in a subject sample, e.g., a blood or urine sample. In some embodiments, diagnosis is confirmed by genetic analysis for the presence of genetic mutations associated with the disease. As used herein, treatment includes treatments with the compositions and methods provided herein to a subject who meets diagnostic criteria of the presence, or absence, of a biomarker, either alone or in combination with a genetic diagnosis, prior to the development of signs or symptoms of the disease. [00382] Enzyme-deficiency diseases that can be treated include non-lysosomal storage disease such as Krabbe disease (galactosylceramidase), phenylketonuria, galactosemia, maple syrup urine disease, mitochondrial disorders, Friedreich ataxia, Zellweger syndrome, adrenoleukodystrophy, Wilson disease, hemochromatosis, ornithine transcarbamylase deficiency, methylmalonic academia, propionic academia, argininosuccinic aciduria, methylmalonic aciduria, type I citrullinemia/argininosuccinate synthetase deficiency, carbamoyl-phosphate synthase 1 deficiency, propionic acidemia, isovaleric acidemia, glutaric academia I, progressive familial intrahepatic cholestasis, types 2 and 3, and lysosomal storage diseases. An enzyme deficiency refers expression and/or activity levels of the enzyme being lower in the subject (e.g., neonatal subject) than normal enzyme expression and/or activity levels, such that the normal functions of the enzyme are not fully carried out in the subject. Routine and pilot newborn screening programs are in place for many enzyme deficiency diseases as treatment is often most effective when started as soon after birth as possible. Screening can be performed on different subject samples depending on the screening test, e.g., urine, dried blood spot. Some preliminary screening tests require follow up analysis to confirm a diagnosis, e.g., genetic sequencing. Such screening and diagnostic methods are well known in the art. Sequencing may indicate a later onset form of the disease that may be managed by screening and delayed intervention at the discretion of a health care professional. As used herein, a subject is considered to have an enzyme deficiency disease if the subject has required signs indicative of the deficiency, e.g., reduced activity level, the presence or absence of a metabolite indicating the presence of disease, or mutations demonstrated by genetic sequencing, prior to the presence of symptoms of the disease, e.g., muscle weakness, failure to thrive. Therefore, administration of the compositions provided herein is understood as treatment of the disease. [00383] Lysosomal storage diseases include any disorder resulting from a defect in lysosome function. Currently, approximately fifty lysosomal storage disorders have been identified, the most well-known of which include Tay-Sachs, Gaucher, and Niemann-Pick disease. The pathogeneses of the diseases are ascribed to the buildup of incomplete degradation products in the lysosome, usually due to loss of protein function. Lysosomal storage diseases are caused by loss-of-function or attenuating variants in the proteins whose normal function is to degrade or coordinate degradation of lysosomal contents. The proteins affiliated with lysosomal storage diseases include enzymes, receptors and other transmembrane proteins (e.g., NPC1), post- translational modifying proteins (e.g., sulfatase), membrane transport proteins, and non- enzymatic cofactors and other soluble proteins (e.g., GM2 ganglioside activator). Thus, lysosomal storage diseases encompass more than those disorders caused by defective enzymes per se, and include any disorder caused by any molecular defect. Thus, as used herein, the term “enzyme” is meant to encompass those other proteins associated with lysosomal storage diseases. [00384] Lysosomal storage diseases are a class of rare diseases that affect the degradation of myriad substrates in the lysosome. Those substrates include sphingolipids, mucopolysaccharides, glycoproteins, glycogen, and oligosaccharides, which can accumulate in the cells of those with disease leading to cell death. Organs affected by lysosomal storage diseases include the central nervous system (CNS), the peripheral nervous system (PNS), lungs, liver, bone, skeletal and cardiac muscle, and the reticuloendothelial system. [00385] Lysosomal storage diseases include sphingolipidoses, a mucopolysaccharidoses, and glycogen storage diseases. In some embodiments, the lysosomal storage disease is any one or more of Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Niemann-Pick disease type A, Niemann-Pick disease type BGM1-gangliosidosis, Sandhoff disease, Tay-Sachs disease, GM2- activator deficiency, GM3-gangliosidosis, metachromatic leukodystrophy, sphingolipid-activator deficiency, Scheie disease, Hurler-Scheie disease, Hurler disease, Hunter disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Morquio syndrome A, Morquio syndrome B, Maroteaux-Lamy disease, Sly disease, MPS IX, and Pompe disease. Enzymes (which include proteins that are not per se catalytic) associated with lysosomal storage diseases include for example any and all hydrolases, α-galactosidase, β-galactosidase, α- glucosidase, β-glucosidase, saposin-C activator, ceramidase, sphingomyelinase, β- hexosaminidase, GM2 activator, GM3 synthase, arylsulfatase, sphingolipid activator, α- iduronidase, iduronidase-2-sulfatase, heparin N-sulfatase, N-acetyl-α-glucosaminidase, α- glucosamide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylgalactosamine-6- sulfate sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, hyaluronidase, and the like. [00386] The nature of the molecular lesion affects the severity of the disease in many cases. Complete loss-of-function tends to be associated with prenatal or neonatal onset, and involves severe symptoms; partial loss-of-function is associated with milder (relatively) and later-onset disease. Generally, only a small percentage of activity needs to be restored to have to correct metabolic defects in deficient cells. Table 8 lists some of the more common lysosomal storage diseases and their associated loss-of-function proteins.
[00387] Table 8: Lysosomal Storage Diseases. [00388] Lysosomal storage diseases can be categorized according to the type of product that accumulates within the defective lysosome. Sphingolipidoses are a class of diseases that affect the metabolism of sphingolipids, which are lipids containing fatty acids linked to aliphatic amino alcohols. The accumulated products of sphingolipidoses include gangliosides (e.g., Tay-Sachs disease), glycolipids (e.g., Fabry’s disease), and glucocerebrosides (e.g., Gaucher’s disease). [00389] Mucopolysaccharidoses are a group of diseases that affect the metabolism of glycosaminoglycans (GAGS or mucopolysaccharides), which are long unbranched chains of repeating disaccharides that help build bone, cartilage, tendons, corneas, skin and connective tissue. The accumulated products of mucopolysaccharidoses include heparan sulfate, dermatan sulfate, keratin sulfate, various forms of chondroitin sulfate, and hyaluronic acid. For example, Morquio syndrome A is due to a defect in the lysosomal enzyme galactose-6-sulfate sulfatase, which results in the lysosomal accumulation of keratin sulfate and chondroitin 6-sulfate. [00390] Glycogen storage diseases result from a cell’s inability to metabolize (make or break- down) glycogen. Glycogen metabolism is moderated by various enzymes or other proteins including glucose-6-phosphatase, acid alpha-glucosidase, glycogen de-branching enzyme, glycogen branching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter, aldolase A, beta- enolase, and glycogen synthase. An exemplar lysosomal storage/glycogen storage disease is Pompe disease, in which defective acid alpha-glucosidase causes glycogen to accumulate in lysosomes. Symptoms include hepatomegaly, muscle weakness, heart failure, and in the case of the infantile variant, death by age two. [00391] Provided herein are methods of treating an enzyme deficiency (e.g., bleeding disorder, inborn error of metabolism, e.g., lysosomal storage disease) in a subject in need thereof (e.g., a neonatal subject). The lysosomal storage disease can be any type of lysosomal storage disease. Examples of lysosomal storage disease is described in more detail elsewhere herein. Such methods can comprise administering any of the nucleic acid constructs described herein (or any of the compositions comprising a nucleic acid construct described herein, including, for example, vectors or lipid nanoparticles) to the subject such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject, for example, wherein the polypeptide of interest is the enzyme in the enzyme deficiency or an enzyme having the same activity as the enzyme in the enzyme deficiency. In some methods, the nucleic acid construct or composition comprising the nucleic acid construct can be administered without a nuclease agent (e.g., if the nucleic acid construct comprises elements needed for expression of polypeptide of interest without integration into a target genomic locus). In some methods, the nucleic acid construct can be administered together with a nuclease agent described herein (e.g., simultaneously or sequentially in any order). The nuclease agent can cleave a nuclease target sequence within a target genomic locus (e.g., target gene), the nucleic acid construct can be inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest can be expressed from the modified target genomic locus (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject). The polypeptide of interest coding sequence can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. In one example, the nuclease agent is a CRISPR/Cas system, and the target gene is ALB (e.g., intron 1 of ALB). In such methods, the guide RNA can bind to the Cas protein and target the Cas protein to the guide RNA target sequence in intron 1 of the ALB gene, the Cas protein can cleave the guide RNA target, the nucleic acid construct can be inserted into the ALB gene to create a modified ALB gene, and polypeptide of interest can be expressed from the modified ALB gene (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject). [00392] Also provided are methods of treating a lysosomal storage disease, for example, in a subject in need thereof (e.g., a neonatal subject). The lysosomal storage disease can be any type of lysosomal storage disease. Examples of lysosomal storage disease are described in more detail elsewhere herein. Such methods can comprise administering any of the nucleic acid constructs described herein (or any of the compositions comprising a nucleic acid construct described herein, including, for example, vectors or lipid nanoparticles) to the subject such that a therapeutically effective level of, for example, polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject, or a polypeptide having the same activity as the polypeptide of interest, wherein the lysosomal storage disease is characterized by loss-of-function of the polypeptide of interest. In some methods, the nucleic acid construct or composition comprising the nucleic acid construct can be administered without a nuclease agent (e.g., if the nucleic acid construct comprises elements needed for expression of polypeptide of interest without integration into a target genomic locus). In some methods, the nucleic acid construct can be administered together with a nuclease agent described herein (e.g., simultaneously or sequentially in any order). The nuclease agent can cleave a nuclease target sequence within a target genomic locus (e.g., target gene), the nucleic acid construct can be inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest can be expressed from the modified target genomic locus (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject). The polypeptide of interest coding sequence can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. In one example, the nuclease agent is a CRISPR/Cas system, and the target gene is ALB (e.g., intron 1 of ALB). In such methods, the guide RNA can bind to the Cas protein and target the Cas protein to the guide RNA target sequence in intron 1 of the ALB gene, the Cas protein can cleave the guide RNA target, the nucleic acid construct can be inserted into the ALB gene to create a modified ALB gene, and polypeptide of interest can be expressed from the modified ALB gene (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject). [00393] Treatment refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of a lysosomal storage disease may comprise alleviating symptoms of the lysosomal storage. Lysosomal storage diseases are described in detail above and can refer to a disorder caused by a missing or defective gene or polypeptide. [00394] Also provided are methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject (e.g., a neonatal subject) in need thereof (e.g., a subject with a lysosomal storage disease characterized by the enzyme deficiency). By preventing is meant the sign or symptom of the enzyme deficiency never becomes present. For example, the methods can prevent or reduce the onset of a sign or symptom of an enzyme deficiency compared to an untreated control subject. The lysosomal storage disease can be any type of lysosomal storage disease. Examples of lysosomal storage disease is described in more detail elsewhere herein. Such methods can comprise administering any of the nucleic acid constructs described herein (or any of the compositions comprising a nucleic acid construct described herein, including, for example, vectors or lipid nanoparticles) to the subject such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject, wherein the polypeptide of interest is the enzyme in the enzyme deficiency. In some methods, the nucleic acid construct or composition comprising the nucleic acid construct can be administered without a nuclease agent (e.g., if the nucleic acid construct comprises elements needed for expression of polypeptide of interest without integration into a target genomic locus). In some methods, the nucleic acid construct can be administered together with a nuclease agent described herein (e.g., simultaneously or sequentially in any order). The nuclease agent can cleave a nuclease target sequence within a target genomic locus (e.g., target gene), the nucleic acid construct can be inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest can be expressed from the modified target genomic locus (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject). The polypeptide of interest coding sequence can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. In one example, the nuclease agent is a CRISPR/Cas system, and the target gene is ALB (e.g., intron 1 of ALB). In such methods, the guide RNA can bind to the Cas protein and target the Cas protein to the guide RNA target sequence in intron 1 of the ALB gene, the Cas protein can cleave the guide RNA target, the nucleic acid construct can be inserted into the ALB gene to create a modified ALB gene, and polypeptide of interest can be expressed from the modified ALB gene (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject [00395] Also provided are methods of preventing or reducing the onset of a sign or symptom of a lysosomal storage disease in a subject (e.g., a neonatal subject) in need thereof. By preventing is meant the sign or symptom of the lysosomal storage disease never becomes present. For example, the methods can prevent or reduce the onset of a sign or symptom of a lysosomal storage disease compared to an untreated control subject. The lysosomal storage disease can be any type of lysosomal storage disease. Examples of lysosomal storage disease is described in more detail elsewhere herein. Such methods can comprise administering any of the nucleic acid constructs described herein (or any of the compositions comprising a nucleic acid construct described herein, including, for example, vectors or lipid nanoparticles) to the subject such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject, wherein the polypeptide of interest is the enzyme in the enzyme deficiency. In some methods, the nucleic acid construct or composition comprising the nucleic acid construct can be administered without a nuclease agent (e.g., if the nucleic acid construct comprises elements needed for expression of polypeptide of interest without integration into a target genomic locus). In some methods, the nucleic acid construct can be administered together with a nuclease agent described herein (e.g., simultaneously or sequentially in any order). The nuclease agent can cleave a nuclease target sequence within a target genomic locus (e.g., target gene), the nucleic acid construct can be inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest can be expressed from the modified target genomic locus (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject). The polypeptide of interest coding sequence can be operably linked to an endogenous promoter at the target genomic locus upon integration into the target genomic locus, or it can be operably linked to an exogenous promoter present in the nucleic acid construct. In one example, the nuclease agent is a CRISPR/Cas system, and the target gene is ALB (e.g., intron 1 of ALB). In such methods, the guide RNA can bind to the Cas protein and target the Cas protein to the guide RNA target sequence in intron 1 of the ALB gene, the Cas protein can cleave the guide RNA target, the nucleic acid construct can be inserted into the ALB gene to create a modified ALB gene, and polypeptide of interest can be expressed from the modified ALB gene (e.g., such that a therapeutically effective level of polypeptide of interest expression or a therapeutically effective level of circulating polypeptide of interest is achieved in the subject [00396] In some methods, a therapeutically effective amount of the nucleic acid construct or the composition comprising the nucleic acid construct or the combination of the nucleic acid construct and the nuclease agent (e.g., CRISPR/Cas system) is administered to the subject. A therapeutically effective amount is an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. See, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding. [00397] Therapeutic or pharmaceutical compositions comprising the compositions disclosed herein can be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J. Pharm. Sci. Technol.52:238-311. In certain embodiments, the pharmaceutical compositions are non-pyrogenic. [00398] The subject (e.g., neonatal subject) in any of the above methods can be from any suitable species, such as a eukaryote or a mammal. A mammal can be, for example, a non-human mammal, a human, a rodent, a rat, a mouse, or a hamster. Other non-human mammals include, for example, non-human primates, e.g., monkeys and apes. The term “non-human” excludes humans. Specific examples of suitable species include, but are not limited to, humans, rodents, mice, rats, and non-human primates. In a specific example, the subject or neonatal subject is a human. [00399] Any target genomic locus capable of expressing a gene can be used in the methods described herein, such as a safe harbor locus (safe harbor gene). Such loci are described in more detail elsewhere herein. In a specific example, the target genomic locus can be an endogenous ALB locus, such as an endogenous human ALB locus. For example, the nucleic acid construct can be genomically integrated in intron 1 of the endogenous ALB locus. Endogenous ALB exon 1 can then splice into the coding sequence for the polypeptide of interest in the nucleic acid construct. [00400] Targeted insertion of the nucleic acid construct comprising the polypeptide of interest coding sequence into a target genomic locus, and particularly an endogenous ALB locus, offers multiple advantages. Such methods result in stable modification to allow for stable, long-term expression of the polypeptide of interest. With respect to the ALB locus, such methods are able to utilize the endogenous ALB promoter and regulatory regions to achieve therapeutically effective levels of expression. For example, the coding sequence for the polypeptide of interest in the nucleic acid construct can comprise a promoterless gene, and the inserted nucleic acid construct can be operably linked to an endogenous promoter in the target genomic locus (e.g., ALB locus). Use of an endogenous promoter is advantageous because it obviates the need for inclusion of a promoter in the nucleic acid construct, allowing packaging of larger transgenes that may not normally package efficiently (e.g., in AAV). Alternatively, the coding sequence in the nucleic acid construct can be operably linked to an exogenous promoter in the nucleic acid construct. Examples of types of promoters that can be used are disclosed elsewhere herein. [00401] Optionally, some or all of the endogenous gene (e.g., endogenous ALB gene) at the target genomic locus can be expressed upon insertion of the coding sequence for the polypeptide of interest from the nucleic acid construct. Alternatively, in some methods, none of the endogenous gene at the target genomic locus is expressed. As one example, the modified target genomic locus (e.g., modified ALB locus) after integration of the nucleic acid construct can encode a chimeric protein comprising an endogenous secretion signal (e.g., albumin secretion signal) and the polypeptide of interest encoded by the nucleic acid construct. In another example, the first intron of an ALB locus can be targeted. The secretion signal peptide of ALB is encoded by exon 1 of the ALB gene. In such a scenario, a promoterless cassette bearing a splice acceptor and the polypeptide of interest coding sequence will support expression and secretion of the polypeptide of interest. Splicing between endogenous ALB exon 1 and the integrated coding sequence for the polypeptide of interest creates a chimeric mRNA and protein including the endogenous ALB sequence encoded by exon 1 operably linked to the polypeptide of interest encoded by the integrated nucleic acid construct. [00402] The nucleic acid construct can be inserted into the target genomic locus by any means, including homologous recombination (HR) and non-homologous end joining (NHEJ) as described elsewhere herein. In a specific example, the nucleic acid construct is inserted by NHEJ (e.g., does not comprise a homology arm and is inserted by NHEJ). [00403] In another specific example, the nucleic acid construct can be inserted via homology- independent targeted integration (e.g., directional homology-independent targeted integration). For example, the coding sequence for the polypeptide of interest in the nucleic acid construct can be flanked on each side by a target site for a nuclease agent (e.g., the same target site as in the target genomic locus, and the same nuclease agent being used to cleave the target site in the target genomic locus). The nuclease agent can then cleave the target sites flanking the polypeptide of interest coding sequence. In a specific example, the nucleic acid construct is delivered AAV-mediated delivery, and cleavage of the target sites flanking the polypeptide of interest coding sequence can remove the inverted terminal repeats (ITRs) of the AAV. Removal of the ITRs can make it easier to assess successful targeting, because presence of the ITRs can hamper sequencing efforts due to the repeated sequences. In some methods, the target site in the target genomic locus (e.g., a gRNA target sequence including the flanking protospacer adjacent motif) is no longer present if the polypeptide of interest coding sequence is inserted into the target genomic locus in the correct orientation but it is reformed if the polypeptide of interest coding sequence is inserted into the target genomic locus in the opposite orientation. This can help ensure that the polypeptide of interest coding sequence is inserted in the correct orientation for expression. [00404] In any of the above methods, the nucleic acid construct encoding the polypeptide of interest can be administered simultaneously with the nuclease agent (e.g., CRISPR/Cas system) or not simultaneously (e.g., sequentially in any combination). For example, in a method comprising administering a composition comprising the nucleic acid construct and a nuclease agent, they can be administered separately. For example, the nucleic acid construct can be administered prior to the nuclease agent, subsequent to the nuclease agent, or at the same time as the nuclease agent. [00405] In one example, the nucleic acid construct is administered about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 1 week prior to administering the nuclease agent. In another example, the nucleic acid construct is administered at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 1 week prior to administering the nuclease agent. In another example, the nucleic acid construct is administered about 4 hours to about 24 hours, about 4 hours to about 12 hours, about 4 hours to about 8 hours, about 8 hours to about 24 hours, about 12 hours to about 24 hours, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 7 days, about 3 days to about 7 days, about 4 days to about 7 days, about 5 days to about 7 days, about 6 days to about 7 days, or about 1 day to about 3 days prior to administering the nuclease agent. [00406] In one example, the nucleic acid construct is administered about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 1 week after administering the nuclease agent. In another example, the nucleic acid construct is administered at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 1 week after administering the nuclease agent. In another example, the nucleic acid construct is administered about 4 hours to about 24 hours, about 4 hours to about 12 hours, about 4 hours to about 8 hours, about 8 hours to about 24 hours, about 12 hours to about 24 hours, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 7 days, about 3 days to about 7 days, about 4 days to about 7 days, about 5 days to about 7 days, about 6 days to about 7 days, or about 1 day to about 3 days after administering the nuclease agent. [00407] Any suitable methods of administering nucleic acid constructs and nuclease agents to cells can be used, particularly methods of administering to the liver, and examples of such methods are described in more detail elsewhere herein. In methods of treatment or in methods of targeting a cell (e.g., neonatal cell) in vivo in a subject (e.g., neonatal subject), the nucleic acid construct can be inserted in particular types of cells in the subject. The method and vehicle for introducing the nucleic acid construct and/or the nuclease agent into the subject can affect which types of cells in the subject are targeted. In some methods, for example, the nucleic acid construct is inserted into a target genomic locus (e.g., an endogenous ALB locus) in liver cells, such as hepatocytes. Methods and vehicles for introducing such constructs and nuclease agents into the subject or neonatal subject (including methods and vehicles that target the liver or hepatocytes, such as lipid nanoparticle-mediated delivery and AAV-mediated delivery (e.g., rAAV8-mediated delivery) and intravenous injection), are disclosed in more detail elsewhere herein. [00408] In any of the above methods, the nucleic acid construct and the nuclease agent (e.g., CRISPR/Cas system) can be administered using any suitable delivery system and known method. The nuclease agent components and nucleic acid construct (e.g., the guide RNA, Cas protein, and nucleic acid construct) can be delivered individually or together in any combination, using the same or different delivery methods as appropriate. [00409] In methods in which a CRISPR/Cas system is used, a guide RNA can be introduced into or administered to a subject or cell, for example, in the form of an RNA (e.g., in vitro transcribed RNA, such as the modified guide RNAs disclosed herein) or in the form of a DNA encoding the guide RNA. When introduced in the form of a DNA, the DNA encoding a guide RNA can be operably linked to a promoter active in the cell or in a cell in the subject. For example, a guide RNA may be delivered via AAV and expressed in vivo under a U6 promoter. Such DNAs can be in one or more expression constructs. For example, such expression constructs can be components of a single nucleic acid molecule. Alternatively, they can be separated in any combination among two or more nucleic acid molecules (i.e., DNAs encoding one or more CRISPR RNAs and DNAs encoding one or more tracrRNAs can be components of a separate nucleic acid molecules). [00410] Likewise, Cas proteins can be introduced into a subject or cell in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternatively, a Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as an RNA (e.g., messenger RNA (mRNA)), such as a modified mRNA as disclosed herein, or DNA). Optionally, the nucleic acid encoding the Cas protein can be codon optimized for efficient translation into protein in a particular cell or organism. For example, the nucleic acid encoding the Cas protein can be modified to substitute codons having a higher frequency of usage in a mammalian cell, a human cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the Cas protein is introduced into a cell or a subject, the Cas protein can be transiently, conditionally, or constitutively expressed in the cell or in a cell in the subject. [00411] In one example, the Cas protein is introduced in the form of an mRNA (e.g., a modified mRNA as disclosed herein), and the guide RNA is introduced in the form of RNA such as a modified gRNA as disclosed herein (e.g., together within the same lipid nanoparticle). Guide RNAs can be modified as disclosed elsewhere herein. Likewise, Cas mRNAs can be modified as disclosed elsewhere herein. [00412] In methods in which a nucleic acid construct is inserted following cleavage by a gene- editing system (e.g., a Cas protein), the gene-editing system (e.g., Cas protein) can cleave the target genomic locus to create a single-strand break (nick) or double-strand break, and the cleaved or nicked locus can be repaired by insertion of the nucleic acid construct via non- homologous end joining (NHEJ)-mediated insertion or homology-directed repair. Optionally, repair with the nucleic acid construct removes or disrupts the guide RNA target sequence(s) so that alleles that have been targeted cannot be re-targeted by the CRISPR/Cas reagents. [00413] As explained in more detail elsewhere herein, the nucleic acid constructs can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), they can be single-stranded or double-stranded, and they can be in linear or circular form. The nucleic acid constructs can be naked nucleic acids or can be delivered by viruses, such as AAV. In a specific example, the nucleic acid construct can be delivered via AAV and can be capable of insertion into the target genomic locus (e.g., a safe harbor gene, an ALB gene, or intron 1 of an ALB gene) by non- homologous end joining (e.g., the nucleic acid construct can be one that does not comprise a homology arm). [00414] Some nucleic acid constructs are capable of insertion by non-homologous end joining. In some cases, such nucleic acid constructs do not comprise a homology arm. For example, such nucleic acid constructs can be inserted into a blunt end double-strand break following cleavage with a Cas protein. In a specific example, the nucleic acid construct can be delivered via AAV and can be capable of insertion by non-homologous end joining (e.g., the nucleic acid construct can be one that does not comprise a homology arm). [00415] In another example, the nucleic acid construct can be inserted via homology- independent targeted integration. For example, the nucleic acid construct can be flanked on each side by a guide RNA target sequence (e.g., the same target site as in the target genomic locus, and the CRISPR/Cas reagent (Cas protein and guide RNA) being used to cleave the target site in the target genomic locus). The Cas protein can then cleave the target sites flanking the nucleic acid insert. In a specific example, the nucleic acid construct is delivered AAV-mediated delivery, and cleavage of the target sites flanking the nucleic acid insert can remove the inverted terminal repeats (ITRs) of the AAV. In some methods, the target site in the target genomic locus (e.g., a guide RNA target sequence including the flanking protospacer adjacent motif) is no longer present if the nucleic acid insert is inserted into the target genomic locus in the correct orientation but it is reformed if the nucleic acid insert is inserted into the target genomic locus in the opposite orientation. [00416] The methods disclosed herein can comprise introducing or administering into a subject or neonatal subject (e.g., an animal or mammal, such as a human) or cell or neonatal cell a nucleic acid construct encoding a polypeptide of interest and optionally a nuclease agent such as CRISPR/Cas reagents, including in the form of nucleic acids (e.g., DNA or RNA), proteins, or nucleic-acid-protein complexes. “Introducing” or “administering” includes presenting to the cell or subject the molecule(s) (e.g., nucleic acid(s) or protein(s)) in such a manner that it gains access to the interior of the cell or to the interior of cells within the subject. The introducing can be accomplished by any means, and two or more of the components (e.g., two of the components, or all of the components) can be introduced into the cell or subject simultaneously or sequentially in any combination. For example, a Cas protein can be introduced into a cell or subject before introduction of a guide RNA, or it can be introduced following introduction of the guide RNA. As another example, a nucleic acid construct can be introduced prior to the introduction of a Cas protein and a guide RNA, or it can be introduced following introduction of the Cas protein and the guide RNA (e.g., the nucleic acid construct can be administered about 1, 2, 3, 4, 8, 12, 24, 36, 48, or 72 hours before or after introduction of the Cas protein and the guide RNA). See, e.g., US 2015/0240263 and US 2015/0110762, each of which is herein incorporated by reference in its entirety for all purposes. In addition, two or more of the components can be introduced into the cell or subject by the same delivery method or different delivery methods. Similarly, two or more of the components can be introduced into a subject by the same route of administration or different routes of administration. [00417] A guide RNA can be introduced into a subject or cell, for example, in the form of an RNA (e.g., in vitro transcribed RNA) or in the form of a DNA encoding the guide RNA. Guide RNAs can be modified as disclosed elsewhere herein. When introduced in the form of a DNA, the DNA encoding a guide RNA can be operably linked to a promoter active in the cell or in a cell in the subject. For example, a guide RNA may be delivered via AAV and expressed in vivo under a U6 promoter. Such DNAs can be in one or more expression constructs. For example, such expression constructs can be components of a single nucleic acid molecule. Alternatively, they can be separated in any combination among two or more nucleic acid molecules (i.e., DNAs encoding one or more CRISPR RNAs and DNAs encoding one or more tracrRNAs can be components of a separate nucleic acid molecules). [00418] Likewise, Cas proteins can be provided in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternatively, a Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as an RNA (e.g., messenger RNA (mRNA)) or DNA. Cas RNAs can be modified as disclosed elsewhere herein. Optionally, the nucleic acid encoding the Cas protein can be codon optimized for efficient translation into protein in a particular cell or organism. For example, the nucleic acid encoding the Cas protein can be modified to substitute codons having a higher frequency of usage in a mammalian cell, a human cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the Cas protein is introduced into a cell or a subject, the Cas protein can be transiently, conditionally, or constitutively expressed in the cell or in a cell in the subject. [00419] Nucleic acids encoding Cas proteins or guide RNAs can be operably linked to a promoter in an expression construct. Expression constructs include any nucleic acid constructs capable of directing expression of a gene or other nucleic acid sequence of interest (e.g., a Cas gene) and which can transfer such a nucleic acid sequence of interest to a target cell. For example, the nucleic acid encoding the Cas protein can be in a vector comprising a DNA encoding one or more gRNAs. Alternatively, it can be in a vector or plasmid that is separate from the vector comprising the DNA encoding one or more gRNAs. Suitable promoters that can be used in an expression construct include promoters active, for example, in one or more of a eukaryotic cell, a human cell, a non-human cell, a mammalian cell, a non-human mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, a pluripotent cell, an embryonic stem (ES) cell, an adult stem cell, a developmentally restricted progenitor cell, an induced pluripotent stem (iPS) cell, or a one-cell stage embryo. For example, a suitable promoter can be active in a liver cell such as a hepatocyte. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Optionally, the promoter can be a bidirectional promoter driving expression of both a Cas protein in one direction and a guide RNA in the other direction. Such bidirectional promoters can consist of (1) a complete, conventional, unidirectional Pol III promoter that contains 3 external control elements: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box; and (2) a second basic Pol III promoter that includes a PSE and a TATA box fused to the 5^ terminus of the DSE in reverse orientation. For example, in the H1 promoter, the DSE is adjacent to the PSE and the TATA box, and the promoter can be rendered bidirectional by creating a hybrid promoter in which transcription in the reverse direction is controlled by appending a PSE and TATA box derived from the U6 promoter. See, e.g., US 2016/0074535, herein incorporated by references in its entirety for all purposes. Use of a bidirectional promoter to express genes encoding a Cas protein and a guide RNA simultaneously allows for the generation of compact expression cassettes to facilitate delivery. In preferred embodiments, promotors are accepted by regulatory authorities for use in humans. In certain embodiments, promotors drive expression in a liver cell. [00420] Molecules (e.g., Cas proteins or guide RNAs or nucleic acids encoding) introduced into the subject or cell can be provided in compositions comprising a carrier increasing the stability of the introduced molecules (e.g., prolonging the period under given conditions of storage (e.g., -20°C, 4°C, or ambient temperature) for which degradation products remain below a threshold, such below 0.5% by weight of the starting nucleic acid or protein; or increasing the stability in vivo). Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, and lipid microtubules. [00421] Various methods and compositions are provided herein to allow for introduction of molecule (e.g., a nucleic acid or protein) into a cell or subject. Methods for introducing molecules into various cell types are known and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods. [00422] Transfection protocols as well as protocols for introducing molecules into cells may vary. Non-limiting transfection methods include chemical-based transfection methods using liposomes; nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2): 456–67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. U.S.A.74 (4):1590–4, and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: W. H. Freeman and Company. pp. 96–97); dendrimers; or cationic polymers such as DEAE-dextran or polyethylenimine. Non- chemical methods include electroporation, sonoporation, and optical transfection. Particle-based transfection includes the use of a gene gun, or magnet-assisted transfection (Bertram (2006) Current Pharmaceutical Biotechnology 7, 277–28). Viral methods can also be used for transfection. [00423] Introduction of nucleic acids or proteins into a cell can also be mediated by electroporation, by intracytoplasmic injection, by viral infection, by adenovirus, by adeno- associated virus, by lentivirus, by retrovirus, by transfection, by lipid-mediated transfection, or by nucleofection. Nucleofection is an improved electroporation technology that enables nucleic acid substrates to be delivered not only to the cytoplasm but also through the nuclear membrane and into the nucleus. In addition, use of nucleofection in the methods disclosed herein typically requires much fewer cells than regular electroporation (e.g., only about 2 million compared with 7 million by regular electroporation). In one example, nucleofection is performed using the LONZA ® NUCLEOFECTOR™ system. [00424] Introduction of molecules (e.g., nucleic acids or proteins) into a cell (e.g., a zygote) can also be accomplished by microinjection. In zygotes (i.e., one-cell stage embryos), microinjection can be into the maternal and/or paternal pronucleus or into the cytoplasm. If the microinjection is into only one pronucleus, the paternal pronucleus is preferable due to its larger size. Microinjection of an mRNA is preferably into the cytoplasm (e.g., to deliver mRNA directly to the translation machinery), while microinjection of a Cas protein or a polynucleotide encoding a Cas protein or encoding an RNA is preferable into the nucleus/pronucleus. Alternatively, microinjection can be carried out by injection into both the nucleus/pronucleus and the cytoplasm: a needle can first be introduced into the nucleus/pronucleus and a first amount can be injected, and while removing the needle from the one-cell stage embryo a second amount can be injected into the cytoplasm. If a Cas protein is injected into the cytoplasm, the Cas protein preferably comprises a nuclear localization signal to ensure delivery to the nucleus/pronucleus. Methods for carrying out microinjection are well known. See, e.g., Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); see also Meyer et al. (2010) Proc. Natl. Acad. Sci. U.S.A.107:15022-15026 and Meyer et al. (2012) Proc. Natl. Acad. Sci. U.S.A.109:9354-9359, each of which is herein incorporated by reference in its entirety for all purposes. [00425] Other methods for introducing molecules (e.g., nucleic acid or proteins) into a cell or subject can include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery, or implantable-device-mediated delivery. As specific examples, a nucleic acid or protein can be introduced into a cell or subject in a carrier such as a poly(lactic acid) (PLA) microsphere, a poly(D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule. Some specific examples of delivery to a subject include hydrodynamic delivery, virus-mediated delivery (e.g., adeno-associated virus (AAV)-mediated delivery), and lipid-nanoparticle-mediated delivery. [00426] Introduction of nucleic acids and proteins into cells or subjects can be accomplished by hydrodynamic delivery (HDD). For gene delivery to parenchymal cells, only essential DNA sequences need to be injected via a selected blood vessel, eliminating safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA is capable of reaching cells in the different tissues accessible to the blood. Hydrodynamic delivery employs the force generated by the rapid injection of a large volume of solution into the incompressible blood in the circulation to overcome the physical barriers of endothelium and cell membranes that prevent large and membrane-impermeable compounds from entering parenchymal cells. In addition to the delivery of DNA, this method is useful for the efficient intracellular delivery of RNA, proteins, and other small compounds in vivo. See, e.g., Bonamassa et al. (2011) Pharm. Res.28(4):694-701, herein incorporated by reference in its entirety for all purposes. [00427] Introduction of nucleic acids can also be accomplished by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses/viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and non- dividing cells. The viruses can integrate into the host genome or alternatively do not integrate into the host genome. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or can be replication-defective (e.g., defective in one or more genes necessary for additional rounds of virion replication and/or packaging). Viruses can cause transient expression or longer-lasting expression. Viral vector may be genetically modified from their wild type counterparts. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some examples, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some examples, the viral vector may have an enhanced transduction efficiency. In some examples, the immune response induced by the virus in a host may be reduced. In some examples, viral genes (such as integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some examples, the viral vector may be replication defective. In some examples, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some examples, the virus may be helper-dependent. For example, the virus may need one or more helper virus to supply viral components (such as viral proteins) required to amplify and package the vectors into viral particles. In such a case, one or more helper components, including one or more vectors encoding the viral components, may be introduced into a host cell or population of host cells along with the vector system described herein. In other examples, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without a helper virus. In some examples, the vector system described herein may also encode the viral components required for virus amplification and packaging. [00428] Exemplary viral titers (e.g., AAV titers) include about 10 12 to about 10 16 vg/mL. Other exemplary viral titers (e.g., AAV titers) include about 10 12 to about 10 16 vg/kg of body weight. [00429] Introduction of nucleic acids and proteins can also be accomplished by lipid nanoparticle (LNP)-mediated delivery. For example, LNP-mediated delivery can be used to deliver a combination of Cas mRNA and guide RNA or a combination of Cas protein and guide RNA. LNP-mediated delivery can be used to deliver a guide RNA in the form of RNA. In a specific example, the guide RNA and the Cas protein are each introduced in the form of RNA via LNP-mediated delivery in the same LNP. As discussed in more detail elsewhere herein, one or more of the RNAs can be modified. For example, guide RNAs can be modified to comprise one or more stabilizing end modifications at the 5’ end and/or the 3’ end. Such modifications can include, for example, one or more phosphorothioate linkages at the 5’ end and/or the 3’ end or one or more 2’-O-methyl modifications at the 5’ end and/or the 3’ end. As another example, Cas mRNA modifications can include substitution with pseudouridine (e.g., fully substituted with pseudouridine), 5’ caps, and polyadenylation. As another example, Cas mRNA modifications can include substitution with N1-methyl-pseudouridine (e.g., fully substituted with N1-methyl- pseudouridine), 5’ caps, and polyadenylation. Other modifications are also contemplated as disclosed elsewhere herein. Delivery through such methods can result in transient Cas expression and/or transient presence of the guide RNA, and the biodegradable lipids improve clearance, improve tolerability, and decrease immunogenicity. Lipid formulations can protect biological molecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles comprising a plurality of lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations which contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time for which nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO 2016/010840 A1 and WO 2017/173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. An exemplary lipid nanoparticle can comprise a cationic lipid and one or more other components. In one example, the other component can comprise a helper lipid such as cholesterol. In another example, the other components can comprise a helper lipid such as cholesterol and a neutral lipid such as DSPC. In another example, the other components can comprise a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033. [00430] The LNP may contain one or more or all of the following: (i) a lipid for encapsulation and for endosomal escape; (ii) a neutral lipid for stabilization; (iii) a helper lipid for stabilization; and (iv) a stealth lipid. See, e.g., Finn et al. (2018) Cell Rep.22(9):2227-2235 and WO 2017/173054 A1, each of which is herein incorporated by reference in its entirety for all purposes. In certain LNPs, the cargo can include a guide RNA or a nucleic acid encoding a guide RNA. In certain LNPs, the cargo can include an mRNA encoding a Cas nuclease, such as Cas9, and a guide RNA or a nucleic acid encoding a guide RNA. In certain LNPs, the cargo can include a nucleic acid construct. In certain LNPs, the cargo can include an mRNA encoding a Cas nuclease, such as Cas9, a guide RNA or a nucleic acid encoding a guide RNA, and a nucleic acid construct. LNPs for use in the methods are described in more detail elsewhere herein. [00431] The mode of delivery can be selected to decrease immunogenicity. For example, a Cas protein and a gRNA may be delivered by different modes (e.g., bi-modal delivery). These different modes may confer different pharmacodynamics or pharmacokinetic properties on the subject delivered molecule (e.g., Cas or nucleic acid encoding, gRNA or nucleic acid encoding, or nucleic acid construct encoding a polypeptide of interest). For example, the different modes can result in different tissue distribution, different half-life, or different temporal distribution. Some modes of delivery (e.g., delivery of a nucleic acid vector that persists in a cell by autonomous replication or genomic integration) result in more persistent expression and presence of the molecule, whereas other modes of delivery are transient and less persistent (e.g., delivery of an RNA or a protein). Delivery of Cas proteins in a more transient manner, for example as mRNA or protein, can ensure that the Cas/gRNA complex is only present and active for a short period of time and can reduce immunogenicity caused by peptides from the bacterially-derived Cas enzyme being displayed on the surface of the cell by MHC molecules. Such transient delivery can also reduce the possibility of off-target modifications. [00432] Administration in vivo can be by any suitable route including, for example, systemic routes of administration such as parenteral administration, e.g., intravenous, subcutaneous, intra- arterial, or intramuscular. In a specific example, administration in vivo is intravenous. [00433] Compositions comprising the guide RNAs and/or Cas proteins (or nucleic acids encoding the guide RNAs and/or Cas proteins) can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation can depend on the route of administration chosen. Pharmaceutically acceptable means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof. In a specific example, the route of administration and/or formulation or chosen for delivery to the liver (e.g., hepatocytes). [00434] The methods disclosed herein can increase polypeptide of interest levels and/or polypeptide of interest activity levels in a cell or neonatal cell or subject or neonatal subject (e.g., circulating, serum, or plasma levels in a subject or neonatal subject) and can comprise measuring polypeptide of interest levels and/or activity levels in a cell or neonatal cell or subject or neonatal subject (e.g., circulating, serum, or plasma levels in a subject or neonatal subject). In one example, the effectiveness of the treatment in a subject can be assessed by measuring serum or plasma polypeptide of interest activity, wherein an increase in the subject’s or neonatal subject’s plasma level and/or activity of polypeptide of interest indicates effectiveness of the treatment. [00435] In some methods, the subject (e.g., neonatal subject) is a subject (e.g., neonatal subject) with a polypeptide of interest deficiency such that expression and/or activity levels of the polypeptide of interest are lower in the subject (e.g., neonatal subject) than normal polypeptide of interest expression and/or activity levels. [00436] In some methods, polypeptide of interest activity and/or expression levels (e.g., plasma or serum levels) in a subject (e.g., neonatal subject) are increased to about or at least about 2%, about or at least about 10%, about or at least about 25%, about or at least about 50%, about or at least about 75%, or at least about 100%, or more, of normal level. In certain embodiments, the level of expression or activity is measured in a cell or tissue in which a sign or symptom of the loss of function is present. For example, when the loss of function results in muscle dysfunction, the level or activity of the polypeptide of interest is measured in a muscle cell. It is understood that depending on the exogenous protein, the level of activity of the exogenous protein may not compare 1:1 with a native protein based on weight. In such embodiment, the relative activity of the exogenous protein and the native protein can be compared. In certain embodiments, the loss of function is nearly complete such that a relative activity cannot be determined. In certain embodiments, the comparison is made to an appropriate control subject. Selection of an appropriate control subject is within the ability of those of skill in the art. In certain embodiments, the level of expression is sufficient to treat at least one sign or symptom resulting from the loss of function of the protein. [00437] In some methods, the method increases expression and/or activity of the polypeptide of interest over the subject’s baseline expression and/or activity (i.e., expression and/or activity prior to administration). In some methods, polypeptide of interest activity and/or expression levels (e.g., plasma or serum levels) in a subject (e.g., neonatal subject) are increased by about or at least about 10%, about or at least about 25%, about or at least about 50%, about or at least about 75%, or about or at least about 100%, or more, as compared to the subject’s polypeptide of interest activity and/or expression levels (e.g., plasma or serum levels) before administration (i.e., the subject’s baseline levels). It is understood that depending on the exogenous protein, the level of activity of the exogenous protein may not compare 1:1 with a native protein based on weight. In such embodiment, the relative activity of the exogenous protein and the native protein can be compared. In certain embodiments, the loss of function is nearly complete such that a relative activity cannot be determined. In certain embodiments, the level of expression is sufficient to treat at least one sign or symptom resulting from the loss of function of the protein. [00438] In some methods, the method increases expression and/or activity of the polypeptide of interest over the cell’s or population’s baseline expression and/or activity (i.e., expression and/or activity prior to administration). In some methods, polypeptide of interest activity and/or expression levels (e.g., protein levels) in a cell or neonatal cell or population of cells or neonatal cells (e.g., liver cells, or hepatocytes) are increased by about or at least about 10%, about or at least about 25%, about or at least about 50%, about or at least about 75%, about or at least about 100%, or more, as compared to the polypeptide of interest activity and/or protein levels before administration. It is understood that depending on the exogenous protein, the level of activity of the exogenous protein may not compare 1:1 with a native protein based on weight. In such embodiment, the relative activity of the exogenous protein and the native protein can be compared. In certain embodiments, the loss of function is nearly complete such that a relative activity cannot be determined. In certain embodiments, the level of expression is sufficient to treat at least one sign or symptom resulting from the loss of function of the protein. [00439] Some methods comprise expressing a therapeutically effective amount of the polypeptide of interest (e.g., achieving a therapeutically effective level of circulating polypeptide of interest activity in an individual). The specific level of expression required depends, for example, on the degree of the loss of function, e.g., partial or complete, and the particular disease or condition to be treated, e.g., what percent of normal activity is required for the deficiency to not manifest signs or symptoms of the disease. Methods to diagnose and monitor diseases and conditions related to loss of function, diseases related to enzyme deficiencies, are known in the art. Some methods comprise achieving polypeptide of interest activity or expression levels of at least about 5% to about 50% of normal or at least about 50% to about 150% of normal. [00440] In a specific example, the activity level of the plasma or serum polypeptide of interest levels in a subject (e.g., neonatal subject) are increased to about 5% to about 200% of normal plasma or serum polypeptide of interest activity levels (e.g., to or about 100% of normal plasma polypeptide of interest levels). [00441] In a specific example, the polypeptide of interest activity levels in a subject (e.g., neonatal subject) are increased to no more than about 300%, no more than about 250%, no more than about 200%, or no more than about 150% of normal polypeptide of interest activity levels. In a specific example, the plasma polypeptide of interest levels in a subject are increased to no more than about 300%, no more than about 250%, no more than about 200%, or no more than about 150% of normal plasma polypeptide of interest levels. [00442] In some methods, the method results in increased expression of the polypeptide of interest in the subject (e.g., neonatal subject) compared to a method comprising administering an episomal expression vector encoding the polypeptide of interest in a control subject. In some methods, the method results in increased serum levels of the polypeptide of interest in the subject (e.g., neonatal subject) compared to a method comprising administering an episomal expression vector encoding the polypeptide of interest to a control subject. [00443] In some methods in which the subject did not express the polypeptide of interest prior to treatment, the method results in expression of the polypeptide of interest at a detectable level above zero, e.g., at a statistically significant level, a clinically relevant level. [00444] Some methods comprise achieving a durable or sustained effect in a human, such as an at least at least 8 weeks, at least 24 weeks, for example, at least 1 year (52 weeks), or optionally at least 2 year effect, and in some embodiments, at least 3 year, at least 4 year, or at least 5 year effect. Some methods comprise achieving the therapeutic effect in a human in a durable and sustained manner, such as an at least 8 weeks, at least 24 weeks, for example, at least 1 year, or optionally at least 2 year effect, and in some embodiments, at least 3 year, at least 4 year, or at least 5 year effect. In some methods, the increased polypeptide of interest activity and/or expression level in a human is stable for at least at least 8 weeks, at least 24 weeks, for example, at least 1 year, optionally at least 2 years, and in some embodiments, at least 3 years, at least 4 years, or at least 5 years. In some methods, a steady-state activity and/or level of polypeptide of interest in a human is achieved by at least 7 days, at least 14 days, or at least 28 days, optionally at least 56 days, at least 80 days, or at least 96 days. In additional methods, the method comprises maintaining polypeptide of interest activity and/or levels after a single dose in a human for at least 8 weeks, at least 16 weeks, or at least 24 week, or in some embodiments at least 1 year, or at least 2 years, optionally at least 3 years, at least 4 years, or at least 5 years. For example, expression of the polypeptide of interest can be sustained in the human subject for at least about 8 weeks, at least about 12 weeks, at least about 24 weeks, in certain embodiments, at least about 1 year, or at least about 2 years after treatment, and in some embodiments, at least 3 years, at least 4 years, or at least 5 years after treatment. Likewise, activity of the polypeptide of interest can be sustained in the human subject for at least about 8 weeks, at least about 12 weeks, at least about 24 weeks, in certain embodiments for at least about 1 year, or at least about 2 years after treatment, and in some embodiments, at least 3 years, at least 4 years, or at least 5 years after treatment. In some methods, expression or activity of the polypeptide of interest is maintained at a level higher than the expression or activity of the polypeptide of interest prior to treatment (i.e., the subject’s baseline). In some methods, expression or activity of the polypeptide of interest is considered sustained if it is maintained at a therapeutically effective level of expression or activity. Relative durations, in other organisms, are understood based, e.g., on life span and developmental stages, are covered within the disclosure above. In some methods, expression or activity of the polypeptide of interest is considered “sustained” if the expression or activity in a human at six months after administration, one year after administration, or two years after administration, the expression or activity is at least 50% of the expression or activity of the peak level of expression or activity measured for that subject. In certain embodiments, at six months, e.g., 24 weeks to 28 weeks, after administration the expression or activity is at least 50%, 55%, 60%, 65%, 70%, 75% or 80% of the expression or activity of the peak level of expression or activity measured for that subject. In certain embodiments, at one year, i.e., about 12 months, e.g., 11-13 months, after administration the expression or activity is at least 50%, 55%, 60%, 65%, 70%, 75% or 80% of the expression or activity of the peak level of expression or activity measured for that subject. In certain embodiments, at two years, i.e., about 24 months, e.g., 23-25 months, after administration the expression or activity is at least 50%, 55%, 60%, 65%, 70%, 75% or 80% of the expression or activity of the peak level of expression or activity measured for that subject. In certain embodiments, at six months after administration the expression or activity is at least 50%, preferably at least 60% of the expression or activity of the peak level of expression or activity measured for that subject. In certain embodiments, at one year after administration the expression or activity is at least 50%, preferably at least 60% of the expression or activity of the peak level of expression or activity measured for that subject. In certain embodiments, at two years after administration the expression or activity is at least 50%, preferably at least 60% of the expression or activity of the peak level of expression or activity measured for that subject. In preferred embodiments, the subject has routine monitoring of expression or activity levels of the polypeptide, e.g., weekly, monthly, particularly early after administration, e.g., within the first six months. Periodic measurements may establish that the effect on expression or activity is sustained at, e.g.6 months after administration, one year after administration, or two years after administration. In some methods in neonatal subjects, the expression of the polypeptide of interest is sustained when the neonatal subject becomes an adult. In some methods, the expression of the polypeptide of interest is sustained for the lifetime of the subject or neonatal subject. [00445] In some methods, the expression or activity of the polypeptide of interest is at least 50% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at 24 weeks after the administering. In some methods, the expression or activity of the polypeptide of interest is at least 50% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at one year after the administering. In some methods, the expression or activity of the polypeptide of interest is at least 60% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at 24 weeks after the administering. In some methods, expression or activity of the polypeptide of interest is at least 50% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at two years after the administering. In some methods, the expression or activity of the polypeptide of interest is at least 60% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at 2 years after the administering. In some methods, the expression or activity of the polypeptide of interest is at least 60% of the expression or activity of the polypeptide at a peak level of expression measured for the human subject at 24 weeks after the administering. [00446] In some methods involving insertion into an ALB locus, the subject’s (e.g., neonatal subject’s) circulating albumin levels or cell’s (e.g., neonatal cell’s) albumin levels are normal. Such methods may comprise maintaining the subject’s (e.g., neonatal subject’s)circulating albumin levels or the cell’s (e.g., neonatal cell’s) albumin levels within ±5%, ±10%, ±15%, ±20%, or ±50% of normal circulating albumin levels or normal albumin levels. In some methods, the subject’s (e.g., neonatal subject’s) or cell’s (e.g., neonatal cell’s) albumin levels are unchanged as compared to the albumin levels of untreated individuals by at least week 4, at least week 8, at least week 12, or at least week 20. In some methods, the subject’s (e.g., neonatal subject’s) or cell’s (e.g., neonatal cell’s) albumin levels transiently drop and then return to normal levels. In particular, the methods may comprise detecting no significant alterations in levels of plasma albumin. [00447] In some methods, the method further comprises assessing preexisting anti-AAV (e.g., anti-AAV8) immunity in a subject prior to administering any of the nucleic acid constructs described herein. For example, such methods could comprise assessing immunogenicity using a total antibody (TAb) immune assay or a neutralizing antibody (NAb) assay. See, e.g., Manno et al. (2006) Nat. Med.12(3):342-347, Kruzik et al. (2019) Mol. Ther. Methods Clin. Dev.14:126- 133, and Weber (2021) Front. Immunol.12:658399, each of which is herein incorporated by reference in its entirety for all purposes. In some embodiments, TAb assays look for antibodies that bind to the AAV vector, whereas NAb assays assess whether the antibodies that are present stop the AAV vector from transducing target cells. With TAb assays, the drug product or an empty capsid can be used to capture the antibodies; NAb assays can require a reporter vector (e.g., a version of the AAV vector encoding luciferase). [00448] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. BRIEF DESCRIPTION OF THE SEQUENCES [00449] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5’ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3’ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. When a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon degenerate variants thereof that encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus. [00450] Table 9. Description of Sequences.
EXAMPLES Example 1. Development of System for Neonatal Insertion into Albumin Locus in Liver [00451] A system for nuclease-mediated insertion (e.g., CRISPR/Cas) of a transgene into a specific locus (e.g., albumin intron 1) was developed to produce durable expression of the transgene, including when administered to neonates. Exemplary components of the system, including those used in subsequent examples, are described in more detail below. Single Guide RNA Design and Selection [00452] The ALB locus was selected as the insertion site for the DNA templates. A list of single guide RNAs (sgRNAs) was generated that target human ALB intron 1. See Table 10. Candidate sgRNAs were synthesized and formulated into lipid nanoparticles (LNPs) with Cas9 mRNA for evaluation in vitro and in vivo. [00453] Table 10. Human ALB Intron 1 Guide RNAs. [00454] LNPs were first screened in primary human hepatocytes (PHH) using a bidirectional nanoluc-encoding AAV insertion template as a reporter. LNPs that supported targeted insertion of nanoluc were identified by measuring nanoluc protein secreted into the supernatant of PHH cultures. Candidates that passed initial PHH screening were then tested for their ability to support in vivo gene insertion. Top candidates from in vivo studies were functionally evaluated for off-target cutting. [00455] LNP-g9860, which is formulated with ALB-targeting sgRNA 9860, described in more detail below, was selected based on supporting robust transgene expression levels across multiple platforms (primary human and non-human primate hepatocytes, ALB humanized mice, and non-human primates), lack of confirmed off-target sites, translation across species, lack of common human SNPs in the target site, low variability of transgene expression within groups, and performance across a dose range. The target site of sgRNA 9860 is conserved in cynomolgus monkeys. LNP-g9860 had no detectable off-target sites in the human genome (targeted amplicon sequencing performed in two lots of primary human hepatocytes at saturating levels of editing failed to validate any locus other than on-target at ALB) and supported transgene expression via insertion in primary human and non-human primate hepatocytes, ALB humanized mice, and non- human primates. LNP-g9860 [00456] LNP-g9860 was developed for use in targeting human ALB intron 1. LNP-g9860 is a lipid nanoparticle that includes a sgRNA of 100 nucleotides in length (g9860) and Cas9- encoding mRNA, each of which is described further below, encapsulated in an LNP comprised of four different lipids. The Cas9 protein, expressed from the Cas9 mRNA, is directed to cleave the DNA when sgRNA 9860 binds to the targeted complementary DNA sequence associated with a PAM. The composition of the LNP is summarized in Table 11. LNP-g9860 comprises four lipids at the following molar ratios: 50 mol% Lipid A, 9 mol% DSPC, 38 mol% cholesterol, and 3 mol% PEG2k-DMG and is formulated in aqueous buffer composed of 50 mM Tris-HCl, 45 mM NaCl, 5% (w/v) sucrose, at pH 7.4. The N:P ratio is about 6, and the gRNA:Cas9 mRNA ratio is about 1:2 by weight. [00457] Table 11. Lipid Nanoparticle (LNP-g9860) Composition. [00458] Single guide RNA. The single guide RNA (sgRNA 9860) used in LNP-g9860 is a 100-mer oligonucleotide containing a 20-nucleotide sequence that is complementary to the target region in intron 1 of the human ALB gene. The target sequence recognized by g9860 is conserved in the cynomolgus monkey mfAlb gene intron 1. The sequence for g9860 is set forth in SEQ ID NOs: 68 and 100. Chemical modifications are incorporated into the 100-mer during synthesis, which include phosphorothioate (PS) linkages at the 5^- and 3^-end of the sgRNA and 2^-O-methyl modifications to some of the sugars of the RNA. [00459] Cas9 mRNA. The Cas9 messenger RNA (mRNA) used in LNP-g9860 is based on the Cas9 protein sequence from Streptococcus pyogenes. The Cas9-encoding mRNA (SEQ ID NO: 1, with a coding sequence (CDS) set forth in SEQ ID NO: 2), is approximately 4400 nucleotides in length. The sequence contains a 5' cap, a 5' untranslated region (UTR), an open reading frame (ORF) encoding the Cas9 protein, a 3' UTR, and a polyA tail. The 5' cap is generated co- transcriptionally by use of a synthetic cap analogue structure, known as anti-reverse cap analogue (ARCA). The uracils in the mRNA sequence have been completely replaced by a modified N 1 methylpseudouridine during the in vitro transcription. The 5^ end of the mRNA has a synthetic cap analog structure. The poly-A tail is approximately 100 nucleotides. LNP-g666 [00460] LNP-g666 was developed for use in targeting mouse Alb intron 1. LNP-g666 is the same as LNP-g9860, except human-albumin-targeting g9860 is replaced with g666, a guide RNA targeting mouse albumin intron 1. The sequence for g666 is set forth in SEQ ID NOS: 166 and 167. rAAV8 Vector [00461] A recombinant AAV8 (rAAV8) vector was developed to carry the DNA insertion templates. The rAAV8 vector carrying the DNA insertion templates is a non-replicating vector that is an AAV-based vector derived from AAV serotype 8. The genome is a single-stranded deoxyribonucleic acid (DNA), comprising inverted terminal repeats (ITR) at each end. The ITRs flank the promoterless insertion template. The AAV ITRs flanking the cassette were derived from AAV2. The DNA insertion templates delivered by rAAV8 vector can be designed as promoterless templates, thus relying on the targeted ALB locus promoter for expression. Example 2. Durable Human FIX Protein Expression After Insertion in Neonatal Mice [00462] To compare episome-mediated expression versus insertion-mediated expression in adult and neonatal mice, and to compare different DNA repair pathways in adult and neonatal mice, we compared hFIX serum levels following administration of a hFIX episome (expression driven by hAAT promoter), a bidirectional hFIX NHEJ insertion template, a hFIX HDR insertion template with homology arms of 500 bp, and a hFIX HDR insertion template with homology arms of 800 bp. See FIG.1. Neonatal C57BL/6 mice were dosed at P0 or P1 with the following: (1) 4 mg/kg of LNP-g666 and 3e9 vg/mouse of rAAV8 with the hFIX-HDR-500 template; (2) 4 mg/kg of LNP-g666 and 3e9 vg/mouse of rAAV8 with the hFIX-HDR-800 template; (3) 4 mg/kg of LNP-g666 and 3e9 vg/mouse of rAAV8 with the hFIX-NHEJ template; or (4) 3e9 vg/mouse of rAAV8 episomal template. Saline-injected mice were used as a negative control. The hFIX coding sequence in the episomal AAV was a codon-optimized sequence encoding wild type human F9. The hFIX coding sequence in the two HDR constructs was the native human F9 coding sequence with the Padua mutation (R338L). Blood was collected and plasma prepared at 1 week, 2 weeks, and 5 weeks post-dosing. hFIX levels were measured by human FIX ELISA. The experiment was then repeated in adult C57BL/6 mice, with the adult mice being dosed with the following: (1) 0.8 mg/kg of LNP-g666 and 2e10 vg/mouse of rAAV8 with the hFIX-HDR-500 template; (2) 0.8 mg/kg of LNP-g666 and 2e10 vg/mouse of rAAV8 with the hFIX-HDR-800 template; (3) 0.8 mg/kg of LNP-g666 and 2e10 vg/mouse of rAAV8 with the hFIX-NHEJ template; or (4) 2e10 vg/mouse of rAAV8 episomal template. Saline- injected mice were used as a negative control. Blood was collected and plasma prepared at 1 week, 2 weeks, and 4 weeks post-dosing. The results are shown in FIGS.2A-2B and Tables 12- 14. Episome-mediated expression was low even at the first time point compared to insertion- mediated expression in neonates and was lost over time in neonates. The opposite was observed in adult mice: episome-mediated expression was higher at the first time point and subsequent time points compared to insertion-mediated expression in adult mice. These results confirmed what was observed in a previous similar experiment (data not shown). In contrast to the results in the neonatal mice, hFIX levels stayed steady in adult mice with both episomal and insertion constructs, with the episomal construct giving the highest expression. See FIGS.2A-2B and Tables 12-14. [00463] Table 12. Human FIX Serum Levels (μg/mL) in Neonatal Mice. [00464] Table 13. Human FIX Serum Levels (μg/mL) in Adult Mice. [00465] Table 14. Human FIX Serum Levels (μg/mL) in Neonatal Mice. [00466] These experiments showed that expression of inserted F9 is durable in neonatal livers, indicating that insertion of F9 templates into the albumin locus can result in durable expression in neonatal subjects. These genome integration provided durable expression that was maintained throughout the experiment in neonatal mice. Example 3. Development of Neonatal Insertion System and Reagents for Treatment of Pompe Disease [00467] A system for nuclease-mediated insertion (e.g., CRISPR/Cas) of an anti-CD63:GAA transgene or an anti-TfR:GAA transgene into a specific locus (e.g., albumin intron 1) was developed to produce durable expression of anti-CD63:GAA or anti-TfR:GAA, including when administered to neonates. [00468] Exemplary components of the system for insertion for anti-CD63:GAA, including those used in subsequent examples, are described in more detail below. See FIGS.3-5. The anti- CD63:GAA DNA template in the working examples described below is brought into the liver by a recombinant AAV8 vector, and the CRISPR/Cas9 RNA components (Cas9 mRNA and sgRNA) are delivered to the liver by LNP-mediated delivery (FIGS.3 and 5). The anti- CD63:GAA protein produced by the liver is targeted to lysosomes in the muscle by targeting CD63, which is a rapidly internalizing protein highly expressed in the muscle. See FIG.4. Single guide RNA, LNP-g9860, Cas9 mRNA, and LNP-g666 design and selection were as described in Example 1. [00469] Exemplary components of the system for anti-TfR:GAA, including those used in subsequent examples, are described in more detail below. See FIGS.11-13. The anti-TfR:GAA DNA templates in the working examples described below are brought into the liver by a recombinant AAV8 vector, and the CRISPR/Cas9 RNA components (Cas9 mRNA and sgRNA) are delivered to the liver by LNP-mediated delivery (FIGS.11 and 13). The anti-TfR:GAA protein produced by the liver is targeted the muscle and CNS by targeting TfR, which is expressed in muscle and on brain endothelial cells. Transcytosis of TfR in these cells enables blood-brain-barrier crossing. See FIG.12. Single guide RNA, LNP-g9860, Cas9 mRNA, and LNP-g666 design and selection were as described in Example 1. DNA Template Design and Selection [00470] We engineered a DNA template for insertion of a nucleic encoding anti-CD63:GAA fusions in which the C-terminus of a single-chain fragment variable (scFv) is fused to the N- terminus of amino acids 70–952 of GAA with a glycine-serine linker. The GAA (70-952) sequence is set forth in SEQ ID NO: 173 and is encoded by the sequence set forth in SEQ ID NO: 174. The DNA template is set forth in SEQ ID NO: 580 and encodes the fusion protein set forth in SEQ ID NO: 579. A splice acceptor site is encoded upstream of the anti-CD63:GAA transgene, and a polyadenylation sequence is encoded downstream of the anti-CD63:GAA transgene. The splice acceptor sequence at the 5’ end of the transgene was derived from mouse Alb exon 2 splice acceptor. The polyadenylation sequence at the 3’ end of the transgene was derived from simian virus 40 (SV40). [00471] We engineered DNA templates for insertion of a nucleic encoding anti-TfR:GAA fusions in which the C-terminus of a single-chain fragment variable (scFv) is fused to the N- terminus of amino acids 70–952 of GAA with a glycine-serine linker. The GAA (70-952) sequence is set forth in SEQ ID NO: 173 and is encoded by the sequence set forth in SEQ ID NO: 174. A splice acceptor site is encoded upstream of the anti-TfR:GAA transgene, and a polyadenylation sequence is encoded downstream of the anti-TfR:GAA transgene. The splice acceptor sequence at the 5’ end of the transgene was derived from mouse Alb exon 2 splice acceptor. The polyadenylation sequence at the 3’ end of the transgene was derived from simian virus 40 (SV40). rAAV8 Vector [00472] A recombinant AAV8 (rAAV8) vector was developed to carry the DNA insertion templates. The rAAV8 vector carrying the anti-CD63:GAA DNA template (REGV044) is a non- replicating vector that is an AAV-based vector derived from AAV serotype 8. The genome is a single-stranded deoxyribonucleic acid (DNA), comprising inverted terminal repeats (ITR) at each end. The ITRs flank the anti-CD63:GAA promoterless insertion template. The AAV ITRs flanking the cassette were derived from AAV2. The anti-CD63:GAA DNA template delivered by rAAV8 vector was designed as a promoterless template, thus relying on the targeted ALB locus promoter for expression. [00473] The rAAV8 vector carrying the anti-TfR:GAA DNA template is a non-replicating vector that is an AAV-based vector derived from AAV serotype 8. The genome is a single- stranded deoxyribonucleic acid (DNA), comprising inverted terminal repeats (ITR) at each end. The ITRs flank the anti-TfR:GAA promoterless insertion template. The AAV ITRs flanking the cassette were derived from AAV2. The anti-TfR:GAA DNA template delivered by rAAV8 vector was designed as a promoterless template, thus relying on the targeted ALB locus promoter for expression. Example 4. Durable Alpha-Glucosidase (GAA) Expression after Insertion of Anti- CD63:GAA DNA Template in Neonatal Mice [00474] We next engineered a DNA template for insertion of a nucleic encoding anti- CD63:GAA fusions in which the C-terminus of an anti-CD63 single-chain fragment variable (scFv) is fused to the N-terminus of GAA with a glycine-serine linker (described above). We tested the anti-CD63:GAA insertion template in a Pompe disease (PD) mouse model, Gaa -/- ;Cd63 hu/hu , where Gaa was replaced by LacZ and the protein-coding region of the Cd63 locus was replaced with its human counterpart. Adult (2-month old) male and female Gaa -/- ;Cd63 hu/hu mice (62.5% C57BL/6, 37.5% 129Sv) were dosed intravenously with the following: (1) 4e12 vg/kg recombinant AAV8 encoding anti-CD63:GAA (REGV042); or (2) 1 mg/kg LNP-g666 and 1.2e13 vg/kg recombinant AAV8 anti-CD63:GAA insertion template (REGV044). REGV042 is an episomal AAV that uses a hSerpina1 enhancer and a mTTR promoter to give hepatocyte- specific expression of anti-CD63:GAA, which further includes a human albumin signal peptide. The anti-CD63:GAA coding sequences were identical in REGV042 and REGV044 and are set forth in SEQ ID NO: 580. Untreated Gaa -/- ;Cd63 hu/hu mice and wild type mice were used as controls. Blood was collected and serum prepared at 7 days, 30 days, 2 months, 3 months, 6 months, and 10 months post-administration, and tissues were collected at 10 months post- administration. Anti-CD63:GAA serum levels were quantified using a plate-based sandwich ELISA that detects the scFv portion of the molecule. Anti-CD63:GAA purified protein was used as a protein standard for quantification. Data are shown in FIG.6 and Tables 15-16. At 10 months post-administration, animals were sacrificed, and glycogen levels were quantified in muscle tissue lysates of the sacrificed animals. Tissues were dissected from mice immediately after sacrifice by CO 2 asphyxiation, snap frozen in liquid nitrogen, and stored at -80°C. Tissues were lysed on a benchtop homogenizer with stainless steel beads in distilled water for glycogen measurements or RIPA buffer for protein analyses. Glycogen analysis lysates were boiled and centrifuged to clear debris. Glycogen measurements were performed fluorometrically with a commercial kit according to manufacturer’s instructions (K646, BioVision, Milpitas, CA, USA). As shown in FIG.7 and Tables 17-19, glycogen was significantly reduced to near wild type levels in both the episomal group and the insertion group in heart, quadricep, and diaphragm in adult mice. [00475] Table 15. Serum Levels of Anti-CD63:GAA in μg/mL in Insertion Adult Group. *Cells without data were due to lost samples post-collection. [00476] Table 16. Serum Levels of Anti-CD63:GAA in μg/mL in Episomal Adult Group. [00477] Table 17. Glycogen Levels in Insertion Adult Group. [00478] Table 18. Glycogen Levels in Episomal Adult Group. [00479] Table 19. Glycogen Levels in Control Adult Groups. *Cells without data were due to experimental error. [00480] Similar experiments were then performed in which neonatal Gaa -/- ;Cd63 hu/hu mice (62.5% C57BL/6, 37.5% 129Sv) were dosed intravenously at P1 with the following: (1) 8.2e12 vg/kg recombinant AAV8 encoding anti-CD63:GAA (REGV042); or (2) 4 mg/kg LNP-g666, and 8.2e12 vg/kg recombinant AAV8 anti-CD63:GAA insertion template (REGV044). Untreated Gaa -/- ;Cd63 hu/hu mice and wild type mice were used as controls. Blood was collected and serum prepared at 7 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 12 months, 13 months, and 15 months, and tissues were collected at 3 months and 15 months post-administration. As shown in FIGS.8A-8B and Tables 20-21, in contrast to what was observed when adult mice were dosed, the serum anti-CD63:GAA levels were stable over the 15-month time course in the insertion group, but the episomal group started out lower and dropped off to below the lower limit of quantification using the serum ELISA assay within 1 month when neonatal mice were dosed. Similarly, as shown in FIG.9A and Table 22, glycogen storage at 3 months was normalized to wild type levels in heart, quadricep, gastrocnemius, and diaphragm in the insertion group, but not in the episomal group. Likewise, as shown in FIG.9B and Table 23, glycogen storage at 15 months was normalized to wild type levels in heart, quadricep, gastrocnemius, and diaphragm in the insertion group, and glycogen storage was partially corrected in CNS tissues in the insertion group but not the episomal group. [00481] Table 20. Serum Anti-CD63:GAA Levels (μg/mL) in Neonatal Mice with Insertion Group. *Mouse sacrificed at 3 months for 3-month glycogen assay **Mouse died
[00483] Table 21. Serum Anti-CD63:GAA Levels (μg/mL) in Neonatal Mice with Episomal Group. *Mouse sacrificed [00485] Table 22. Glycogen Levels (μg/mg Tissue) in Neonatal Mice.
[00486] Table 23. Glycogen Levels (μg/mg Tissue) in Neonatal Mice. [00487] To assess whether the improved glycogen reduction observed with the insertion template in neonatal mice translated into improved muscle function, the mice were tested on grip strength apparatuses at 15 months post-administration. Limb grip strength was measured with a force meter (Columbus Instruments, Columbus, OH, USA). All tests were performed in triplicate. Mice treated with the insertion template showed significantly improved performance compared to mice treated with the episomal construct on the grip strength test. In fact, the grip strength in the insertion group tracked closely with that of wild type mice at 15 months post- treatment, whereas there was no difference in the grip strength in the episomal group tracked compared to the untreated group. See FIG.10 and Table 24. These results show that, in neonatal mice, the insertion approach shows vastly improved durability of expression compared to the episomal approach, and better substrate reduction, indicating that insertion is the superior approach for pediatric indications. [00488] Table 24. Grip Strength (Newtons) in Neonatal Mice. [00489] An experiment was performed in which 4-month old Gaa -/- ;Alb hu/hu mice (n=3) were dosed intravenously with 7.5e10 vg/mouse recombinant AAV8 anti-CD63:GAA insertion template (REGV044) and 1 mg/kg LNP-g9860 in order to validate that anti-CD63:GAA can be inserted into mice humanized for albumin using human albumin gRNA. Blood was collected and serum prepared at 7 days, 14 days, 35 days, and 60 days post-administration. GAA serum levels up to ~3 μg/mL were observed and were maintained over the time course (data not shown), confirming that anti-CD63:GAA can be inserted into mice humanized for albumin using human albumin gRNA. [00490] In summary, the combination of the highly precise and targeted CRISPR/Cas9 technology delivered by LNP and the anti-CD63:GAA DNA template delivered by the selected rAAV8 vector allows for long-term expression of anti-CD63:GAA protein from hepatocytes and delivery to muscle cells affected in PD, potentially providing a life-long effective treatment to PD patients, including neonatal patients. [00491] These results show that, in neonatal mice, the insertion approach shows vastly improved durability of expression compared to the episomal approach, indicating that insertion is the superior approach in neonatal subjects. Example 5. Durable Alpha-Glucosidase (GAA) Expression after Insertion of Anti- TfR:GAA DNA Template in Neonatal Mice [00492] Anti-human transferrin receptor (hTfR) antibodies were generated and screened for the ability to bind hTfR and for lack of strong blocking of human transferrin-hTfR binding. Based on this initial analysis, 32 variable sequences were chosen. See Table 25. [00493] Table 25. Domains in Anti-hTfR Antibodies, Antigen-binding Fragments (e.g., Fabs) or scFv Molecules in Fusion Proteins. 31874B HCVR (VH) Nucleotide Sequence GGC CCC GG C CCG C CC C (S Q NO: 6) HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence 31863B HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence 69348 HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence 69340 HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence 69331 HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence 69332 HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence 69326 HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 69329 HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 69323 (REGN16816 anti-hTfR scFv:hGAA) HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence 69305 HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 69307 (REGN16817 anti-hTfR scFv:hGAA) HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence Q ( Q ) LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence Q ( Q ) 12795B HCVR (V H ) Nucleotide Sequence ( Q ) HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 12798B (REGN17078 Fab; REGN17072 scFv; REGN16818 anti-hTfR scFv:hGAA) HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence QQ ( Q ) 12799B (REGN17079 Fab; REGN17073 scFv; REGN16819 anti-hTfR scFv:hGAA) HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 12802B (REGN16820 anti-hTfR scFv:hGAA) HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 12812B (REGN16821 anti-hTfR scFv:hGAA) HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence QQ ( Q ) 12816B HCVR (V H ) Amino Acid Sequence ( Q ) LCVR (VL) Nucleotide Sequence LCVR (VL) Amino Acid Sequence HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence QQ ( Q ) HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence QQ ( Q ) HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence ( ) HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence ( Q ) LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence ( ) HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 12839B (REGN17080 Fab; REGN17074 scFv; REGN16822 anti-hTfR scFv:hGAA) HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 12841B (REGN16823 anti-hTfR scFv:hGAA) HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence 12850B (REGN16828 anti-hTfR scFv:hGAA) HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (V L ) Amino Acid Sequence HCVR (V H ) Nucleotide Sequence HCVR (V H ) Amino Acid Sequence LCVR (VL) Nucleotide Sequence LCVR (VL) Amino Acid Sequence HCVR (VH) Nucleotide Sequence HCVR (VH) Amino Acid Sequence LCVR (V L ) Nucleotide Sequence LCVR (VL) Amino Acid Sequence LCDR3: QKYNSVPLT (SEQ ID NO: 535) [00494] Table 26. Anti-hTfR scFv Molecules in Fusion Proteins. Anti-TfR scFV:GAA Sequences: (SEQ ID NO: 184)
VSWC (SEQ ID NO: 185) (SEQ ID NO: 570) (SEQ ID NO: 186) (SEQ ID NO: 187) (SEQ ID NO: 188) (SEQ ID NO: 189) (SEQ ID NO: 190) (SEQ ID NO: 191) Q (SEQ ID NO: 192) (SEQ ID NO: 193) Q (SEQ ID NO: 571) Q (SEQ ID NO: 194) (SEQ ID NO: 572) (SEQ ID NO: 195)
(SEQ ID NO: 196) (SEQ ID NO: 573) (SEQ ID NO: 197)
(SEQ ID NO: 198) (SEQ ID NO: 199) (SEQ ID NO: 200) Q (SEQ ID NO: 201) VSWC (SEQ ID NO: 202) (25) 69307 (REGN16817) (SEQ ID NO: 204) (SEQ ID NO: 205) (SEQ ID NO: 206) Q (SEQ ID NO: 207) (SEQ ID NO: 208) (30) 69332
(SEQ ID NO: 209) (SEQ ID NO: 210) (SEQ ID NO: 211); (1)
Q (SEQ ID NO: 212; optionally lacking the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 596) sequence); QQ Q (SEQ ID NO: 213; optionally lacking the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 596) sequence);
[00495] In order to validate the anti-human TfR antibodies that were screened for binding in vitro, we performed in vivo mouse studies in Tfrc hum/hum knock-in mice to evaluate blood-brain- barrier (BBB) crossing. Eleven clones that had mature hGAA protein in brain homogenate detected by western blot were selected from this first screen of 31 antibodies. [00496] GAA fusions by hydrodynamic delivery (HDD). Three-month-old human TFRC knock-in mice were injected with DNA plasmids expressing the various anti-hTfR antibodies in the anti-hTfRscfv:2xG4S:hGAA format under the liver-specific mouse TTR promoter. Mice received 50 μg of DNA in 0.9% sterile saline diluted to 10% of the mouse’s body weight (0.1 mL/g body weight).48 hours post-injection, tissues were dissected from mice immediately after sacrifice by CO2 asphyxiation, snap frozen in liquid nitrogen, and stored at -80 o C. [00497] Tissue lysates were prepared by lysis in RIPA buffer with protease inhibitors (1861282, Thermo Fisher, Waltham, MA, USA). Tissue lysates were homogenized with a bead homogenizer (FastPrep5, MP Biomedicals, Santa Ana, CA, USA). Cells or tissue lysates were run on SDS-PAGE gels using the Novex system (LifeTech Thermo, XPO4200BOX, LC2675, LC3675, LC2676). Gels were transferred to low-fluorescence polyvinylidene fluoridev (PVDF) membrane (IPFL07810, LI-COR, Lincoln, NE, USA) and stained with Revert 700 Total Protein Stain (TPS; 926-11010 LI-COR, Lincoln, NE, USA), followed by blocking with Odyssey blocking buffer (927-500000, LI-COR, Lincoln, NE, USA) in Tris buffer saline with 0.1% Tween 20 and staining with antibodies against GAA (ab137068, Abcam, Cambridge, MA, USA), or anti-GAPDH (ab9484, Abcam, Cambridge, MA, USA) and the appropriate secondary (926-32213 or 925-68070, LI-COR, Lincoln, NE, USA). Blots were imaged with a LI-COR Odyssey CLx. [00498] Protein band intensity was quantified in LI-COR Image Studio software. The quantification of the mature 77 kDa GAA band for each sample was determined by first normalizing to the lane’s TPS signal, then normalizing to GAA levels in the serum (loading control and liver expression control, respectively). Values were then compared to the positive control group anti-mouse TfRscfv:hGAA in Wt mice, and negative control group anti- mTfRscfv:hGAA in Tfrc hum/hum mice (FIGS.14A-14C, Table 27). The 8D3 scFv (anti-mouse TfR scFv) has the heavy chain amino acid sequence:
[00499] Table 27. Quantification of mature hGAA protein in brain homogenate from mice treated HDD with anti-hTfRscfv:hGAA plasmids. [00500] Data were quantified from western blot as arbitrary units (FIGS.14A-14C). All values are mean ± SD, n=3-6 per group. One Way ANOVA vs. negative control anti- mTfRscfv:hGAA in Tfrc hum/hum mice; *p<0.05; **p<0.005; ***p<0.0001. [00501] Capillary depletion of brain samples following HDD of anti-hTfRscfv:hGAA plasmids. Selected anti-hTfRscfv:hGAA from Table 27 were tested in a secondary screen in Tfrc hum mice to determine whether hGAA was present in the brain parenchyma, and not trapped in the BBB endothelial cells. We selected four scFvs (12799, 12839, 12843, and 12847) from this screen based on mature hGAA in the parenchyma fraction on western blot, as well as high affinity to cynomolgus TfR. [00502] Three-month-old animals were treated HDD as detailed above.48 hours post- injection, mice were perfused with 30 mL 0.9% saline immediately after sacrifice by CO 2 asphyxiation. A 2 mm coronal slice of cerebrum was taken between bregma and -2 mm bregma and placed in 700 ^L physiological buffer (10 mM HEPES, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 10 mM D-glucose in 0.9% saline pH 7.4) on ice. Brain slices were gently homogenized on ice with a glass dounce homogenizer. An equivalent volume of 26% dextran (MW 70,000 Da) in physiological buffer was added (final 13% dextran) and homogenized 10 more strokes. Parenchyma (supernatant) and endothelial (pellet) fractions were separated by centrifugation at 5,400g for 15 min at 4 o C. Anti-hGAA western blot was performed on fractions as detailed above (FIG.15, Table 28). Blots were also probed with anti-CD31 endothelial marker (Abcam ab182982). [00503] Table 28. Quantification of mature hGAA protein in brain parenchyma fractions and BBB endothelial fractions of mice treated HDD with anti-hTfRscfv:hGAA plasmids. [00504] hGAA protein was quantified from western blot as arbitrary units (FIG.15). n=1 per group. Affinity to cynomolgus macaque TfR Luminex data, calculated as percent of binding to hTfR: (^^^^^^^^^^^^^ ^ ^^^^^^^^^^^^^^^ ^^^^ [00505] Table 29. Quantification of hGAA protein in quadricep of mice treated HDD with anti-hTfRscfv:hGAA plasmids. [00506] Data were quantified from western blot as arbitrary units (FIG.15). All values are mean ± SD, n=2-4 per group. [00507] Capillary depletion of mouse brain samples following liver-depot AAV8 anti- hTfRscfv:hGAA treatment. To confirm our HDD screen findings in a more long-term treatment model, we treated Tfrc hum mice with selected anti-hTfRscfv:GAA delivered as episomal liver depot AAV8 anti-hTfRscfv:GAA under the TTR promoter. We found that all 4 anti-hTfRscfv:GAA delivered mature hGAA to the brain parenchyma when delivered as AAV8. [00508] AAV production and in vivo transduction. Recombinant AAV8 (AAV2/8) was produced in HEK293 cells. Cells were transfected with three plasmids encoding adenovirus helper genes, AAV8 rep and cap genes, and recombinant AAV genomes containing transgenes flanked by AAV2 inverted terminal repeats (ITRs). On day 5, cells and medium were collected, centrifuged, and processed for AAV purification. Cell pellets were lysed by freeze-thaw and cleared by centrifugation. Processed cell lysates and medium were overlaid onto iodixanol gradients columns and centrifuged in an ultracentrifuge. Virus fractions were removed from the interface between the 40% and 60% iodixanol solutions and exchanged into 1xPBS with desalting columns. AAV vg were quantified by ddPCR. AAVs were diluted in PBS + 0.001% F- 68 Pluronic immediately prior to injection. Three-month-old Tfrc hum mice were dosed with 3e12 vg/kg body weight in a volume of ~100 ^L. Mice were sacrificed 4 weeks post injection and capillary depletion and western blotting were performed as described above (FIG.16, Table 30). [00509] Table 30. Quantification of mature hGAA protein in brain parenchyma fractions and BBB endothelial fractions of mice treated with liver-depot AAV8 anti- hTfRscfv:hGAA. [00510] Data were quantified from western blot as arbitrary units (FIG.16). n=1 per group. [00511] Rescue of glycogen storage phenotype in Gaa -/- /Tfrc hum mice with AAV8 episomal liver depot anti-hTfRscfv:GAA. We tested four of the anti-hTfRscfv:GAA from the above experiment in Pompe disease model mice to determine whether hTfRscfv:GAA rescued the glycogen storage phenotype. We found that all four (12839, 12843, 12847, 12799) normalized glycogen to Wt levels. [00512] AAV production and in vivo transduction were performed as above. Three-month-old Gaa -/- /Tfrc hum mice were dosed with 2e12 vg/kg AAV8. Tissues were harvested 4 weeks post- injection and flash-frozen as above. hGAA Western blot was performed as above (FIG.17, Table 31). [00513] Glycogen quantification (Table 32, FIGS.18A-18C). Tissues were dissected from mice immediately after sacrifice by CO 2 asphyxiation, snap frozen in liquid nitrogen, and stored at -80 o C. Tissues were lysed on a benchtop homogenizer with stainless steel beads in distilled water for glycogen measurements or RIPA buffer for protein analyses. Glycogen analysis lysates were boiled and centrifuged to clear debris. Glycogen measurements were performed fluorometrically with a commercial kit according to manufacturer’s instructions (K646, BioVision, Milpitas, CA, USA). All groups had normal iron homeostasis at 4 weeks post- injection (serum iron, TIBC, hepcidin, tissue iron, tissue transferrin). [00514] Table 31. Quantification of hGAA protein in tissues of Gaa -/- /Tfrc hum mice treated with liver-depot AAV8 anti-hTfRscfv:hGAA. [00515] Data were quantified from western blot as arbitrary units (FIG.17). All values are mean ± SD, n=1-3 per group. *Total hGAA protein; **Mature hGAA protein. [00516] Table 32. Quantification of glycogen in tissues of Gaa -/- /Tfrc hum mice treated with liver-depot AAV8 anti-hTfRscfv:hGAA. [00517] All values are glycogen μg/mg tissue, mean ± SD, n=3-4 per group. One Way ANOVA *p<0.0001 vs. Gaa -/- Untreated group. [00518] Rescue of glycogen storage in brain and muscle in Gaa -/- /Tfrc hum mice with AAV8 episomal liver depot anti-hTfRscfv:GAA. We tested three selected anti-hTfRscfv:GAA (12799, 12843, and 12847) in Pompe disease model mice to determine whether hTfRscfv:GAA rescued the glycogen storage phenotype. In this experiment, we performed histology on brain and muscle sections to visualize glycogen in the tissues. We found that all three selected anti- hTfRscfv:GAA reduced glycogen staining in the brain and muscle. We selected 12847scfv:GAA for further analysis based on these data. [00519] AAV production and in vivo transduction were performed as above. Three-month old Gaa -/- /Tfrc hum mice were dosed with 4e11 vg/kg AAV8.4 weeks post-injection, tissues were frozen for glycogen analysis as above (Table 33). For histology, animals were perfused with saline (0.9% NaCl), and tissues were drop-fixed overnight in 10% Normal Buffered Formalin. Tissues were washed 3x in PBS and stored in PBS/0.01% sodium azide until embedding. Tissues were embedded in paraffin and 5um sections were cut from brain (coronal, -2mm bregma) and quadricep (fiber cross-section). Sections were stained with Periodic Acid-Schiff and Hematoxylin using standard protocols (FIGS.19A-19D). [00520] Table 33. Quantification of glycogen in tissues of Gaa -/- /Tfrc hum mice treated with liver-depot AAV8 anti-hTfRscfv:hGAA. [00521] All values are glycogen μg/mg tissue, mean ± SD, n=5-8 per group. One Way ANOVA *p<0.0001 vs. Gaa -/- Untreated group. [00522] Insertion of anti-hTfR 12847scfv:GAA in Gaa -/- /Tfrc hum mice. We tested the selected anti-hTfR 12847scfv:GAA in Pompe disease model mice by albumin insertion to determine whether we could replicate the results we saw with episomal AAV8 liver depot expression. Albumin insertion of 12847scfv:GAA delivered mature hGAA protein to the brain and muscle, and rescued the glycogen storage phenotype in Gaa -/- /Tfrc hum mice. These data were produced with the native 12847scfv:GAA sequence that is not optimized. [00523] We compared 12847scfv:GAA to the muscle-targeted anti-hCD63scfv:GAA in Gaa -/- /Cd63 hum mice. In this particular experiment, the expression of anti-hCD63scfv:GAA was lower than usual and does not deliver as much GAA protein to the muscle nor normalize glycogen as it usually does. This may make it appear that anti-hCD63scfv:GAA is less effective than 12847scfv:GAA in the muscle but in most experiments we found them to be comparable in the muscle. [00524] AAV production. A promoterless AAV genome plasmid was created with the 12847scfv:GAA sequence and the mouse albumin exon 1 splice acceptor site at the 3’ end. Recombinant AAV8 (AAV2/8) was produced in HEK293 cells. Cells were transfected with three plasmids encoding adenovirus helper genes, AAV8 rep and cap genes, and recombinant AAV genomes containing transgenes flanked by AAV2 inverted terminal repeats (ITRs). On day 5, cells and medium were collected, centrifuged, and processed for AAV purification. Cell pellets were lysed by freeze-thaw and cleared by centrifugation. Processed cell lysates and medium were overlaid onto iodixanol gradients columns and centrifuged in an ultracentrifuge. Virus fractions were removed from the interface between the 40% and 60% iodixanol solutions and exchanged into 1xPBS with desalting columns. AAV vg were quantified by ddPCR. [00525] In vivo CRISPR/Cas9 insertion into the albumin locus.3-month old Gaa -/- /Tfrc hum mice were dosed via tail vein injection with 3e12 vg/kg AAV812847scfv:GAA and 3 mg/kg LNP G666/Cas9 mRNA diluted in PBS + 0.001% F-68 Pluronic. Mice were sacrificed 3 weeks post injection. Negative control mice received insertion AAV8 without LNP. Positive control mice were dosed with 4e11 vg/kg episomal liver depot AAV812847scfv:GAA under the TTR promoter (phenotype rescue data previously shown). Tissues were dissected from mice immediately after sacrifice by CO 2 asphyxiation, snap frozen in liquid nitrogen, and stored at - 80 o C. Blood was collected from mice by cardiac puncture immediately following CO2 asphyxiation and serum was separated using serum separator tubes (BD Biosciences, 365967). [00526] Table 34. Treatment Groups and Controls. [00527] Western blot (Table 35, FIG.20A). Tissue lysates were prepared by lysis in RIPA buffer with protease inhibitors (1861282, Thermo Fisher, Waltham, MA, USA). Tissue lysates were homogenized with a bead homogenizer (FastPrep5, MP Biomedicals, Santa Ana, CA, USA). Cells or tissue lysates were run on SDS-PAGE gels using the Novex system (LifeTech Thermo, XPO4200BOX, LC2675, LC3675, LC2676). Gels were transferred to low-fluorescence polyvinylidene fluoridev (PVDF) membrane (IPFL07810, LI-COR, Lincoln, NE, USA) and stained with Revert 700 Total Protein Stain (TPS; 926-11010 LI-COR, Lincoln, NE, USA), followed by blocking with Odyssey blocking buffer (927-500000, LI-COR, Lincoln, NE, USA) in Tris buffer saline with 0.1% Tween 20 and staining with antibodies against GAA (ab137068, Abcam, Cambridge, MA, USA), or anti-GAPDH (ab9484, Abcam, Cambridge, MA, USA) and the appropriate secondary (926-32213 or 925-68070, LI-COR, Lincoln, NE, USA). Blots were imaged with a LI-COR Odyssey CLx. [00528] Protein band intensity was quantified in LI-COR Image Studio software. The quantification of the mature 77 kDa GAA band for each sample was determined by normalizing to the lane’s TPS signal (loading control). [00529] Glycogen quantification (Table 36, FIG.20B). Tissues were dissected from mice immediately after sacrifice by CO 2 asphyxiation, snap frozen in liquid nitrogen, and stored at - 80 o C. Tissues were lysed on a benchtop homogenizer with stainless steel beads in distilled water for glycogen measurements or RIPA buffer for protein analyses. Glycogen analysis lysates were boiled and centrifuged to clear debris. Glycogen measurements were performed fluorometrically with a commercial kit according to manufacturer’s instructions (K646, BioVision, Milpitas, CA, USA). [00530] Table 35. Quantification of hGAA protein in tissues of Gaa -/- /Tfrc hum mice treated with insertion anti-hTfR 12847scfv:hGAA. [00531] All values are arbitrary units, mean ± SD, n=3-8 per group. One Way ANOVA *p<0.05 vs. Gaa -/- episomal AAV8 TTR 12847scfv:GAA group; §§ p<0.001 vs. AAV only negative control group. [00532] Table 36. Quantification of glycogen in tissues of Gaa -/- /Tfrc hum mice treated with insertion anti-hTfR 12847scfv:hGAA. [00533] All values are glycogen μg/mg tissue, mean ± SD, n=3-8 per group. One Way ANOVA *p<0.01 vs. Gaa -/- /Cd63 hum untreated group; **p<0.001 vs. Gaa -/- /Cd63 hum untreated group; ***p<0.0001 vs. Gaa -/- /Tfrc hum untreated group; § non-significant vs. Wt untreated group. [00534] Similar experiments are then performed in which neonatal Gaa -/- ;Tfrc hu/hu mice are dosed intravenously at P1 with the following: (1) recombinant AAV8 encoding anti-TfR:GAA; or (2) LNP-g666 and recombinant AAV8 anti-TfR:GAA insertion template. Untreated Gaa -/- ;Tfrc hu/hu mice and wild type mice are used as controls. Blood is collected and serum prepared at various time points post-administrations, and tissues are collected at various time points post- administration. Serum anti-TfR:GAA levels and glycogen levels in various muscle and CNS tissues are measured over the time course. [00535] To assess whether glycogen reduction translates into improved muscle function, the mice are tested on grip strength apparatuses at a time point post-administration. Limb grip strength is measured with a force meter (Columbus Instruments, Columbus, OH, USA). All tests are performed in triplicate. [00536] In summary, the combination of the highly precise and targeted CRISPR/Cas9 technology delivered by LNP and the anti-TfR:GAA DNA template delivered by the selected rAAV8 vector allows for long-term expression of anti-TfR:GAA protein from hepatocytes and delivery to muscle cells and CNS cells affected in PD, potentially providing a life-long effective treatment to PD patients, including neonatal patients. [00537] Table 37. Additional GAA sequences.
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