BOBO TIERRA (US)
| WO2013149237A1 | 2013-10-03 |
| EP3611186A1 | 2020-02-19 |
BISIO HUGO, CHAABENE ROUAA BEN, SABITZKI RICARDA, MACO BOHUMIL, MARQ JEAN BAPTISTE, GILBERGER TIM-WOLF, SPIELMANN TOBIAS, SOLDATI-: "The ZIP Code of Vesicle Trafficking in Apicomplexa: SEC1/Munc18 and SNARE Proteins", MBIO, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 11, no. 5, 27 October 2020 (2020-10-27), US , XP093034094, ISSN: 2161-2129, DOI: 10.1128/mBio.02092-20
BOBO, T. A.; ROBINSON, MICHAEL; SOKOLSKI-PAPKOV, MARINA; FU, HAIYAN: "AAV Gene Therapy for Treating MPS IIIC: Facilitate by-Stander Effects by EV-mRNA Cargo", MOLECULAR THERAPY, ELSEVIER INC., US, vol. 30, no. 4S1, 31 March 2022 (2022-03-31), US , pages 120 - 121, XP009543546, ISSN: 1525-0016
| We Claim: 1. An expression vector comprising a polynucleotide encoding a nucleic acid of interest operably linked to an extracellular vesicle-targeting zip code sequence. 2. The expression vector of claim 1, wherein the extracellular vesicle-targeting zip code sequence is linked to the 3’ end of the polynucleotide. 3. The expression vector of claim 1 or 2, wherein the polynucleotide is operably linked to a promoter and a poly(A) signal and the extracellular vesicle-targeting zip code sequence is located between the polynucleotide and the poly(A) signal. 4. The expression vector of any one of claims 1-3, wherein the extracellular vesicle- targeting zip code sequence comprises the nucleotide sequence of SEQ ID NO:1. 5. The expression vector of any one of claims 1-4, wherein the expression vector is a plasmid vector. 6. The expression vector of any one of claims 1-4, wherein the expression vector is a viral vector. 7. The expression vector of claim 6, wherein the expression vector is an adeno-associated virus vector. 8. The expression vector of claim 7, wherein the adeno-associated virus comprises a serotype selected from the group consisting of AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and a chimeric AAV vector. 9. The expression vector of claim 7 or 8, wherein the expression vector is a self- complementary AAV vector. 10. The expression vector of any one of claims 1-9, wherein the nucleic acid of interest encodes a therapeutic protein. 11. The expression vector of claim 10, wherein the therapeutic protein is a secreted protein. 12. The expression vector of claim 10, wherein the therapeutic protein is a non-secreted protein. 13. The expression vector of claim 10, wherein the therapeutic protein is heparan alpha- glucosaminide N-acetyltransferase (HGSNAT). 14. The expression vector of claim 10, wherein the therapeutic protein is survival motor neuron 1 (SMN1). 15. The expression vector of any one of claims 1-9, wherein the nucleic acid of interest encodes a functional nucleic acid. 16. A virus particle comprising the expression vector of any one of claims 1-15. 17. The virus particle of claim 16, wherein the virus particle is an AAV particle, an adenovirus particle, a herpesvirus particle, or a baculovirus particle. 18. A pharmaceutical composition comprising the expression vector of any one of claims 1- 15 or the virus particle of claim 16 or 17 and a pharmaceutically acceptable carrier. 19. A method of delivering a nucleic acid of interest to bystander cells in a subject, comprising administering to the subject an effective amount of the expression vector of any one of claims 1-15, the virus particle of claim 16 or 17, or the pharmaceutical composition of claim 18, thereby forming extracellular vesicles comprising the nucleic acid of interest and delivering the nucleic acid of interest to bystander cells. 20. A method of treating a disorder treatable by expression of a nucleic acid of interest in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the expression vector of any one of claims 1-15, the virus particle of claim 16 or 17, or the pharmaceutical composition of claim 18, thereby treating the disorder. 21. The method of claim 20, wherein the nucleic acid of interest encodes a therapeutic protein. 22. The method of claim 21, wherein the therapeutic protein is a secreted protein. 23. The method of claim 21, wherein the therapeutic protein is a non-secreted protein. 24. The method of claim 21, wherein the therapeutic protein is HGSNAT and the disorder is mucopolysaccharidosis IIIC. 25. The method of claim 21, wherein the therapeutic protein is SMN1 and the disorder is spinal muscle atrophy. 26. The method of claim 20, wherein the nucleic acid of interest encodes a functional nucleic acid. 27. The method of any one of claims 19-26, wherein the subject is a human subject. |
Table 1 [0080] The AAV viral vectors of the invention may include a recombinant AAV vector genome. A “recombinant AAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA) that comprises at least one inverted terminal repeat (e.g., one, two or three inverted terminal repeats) and one or more heterologous nucleotide sequences. rAAV vectors generally retain the 145 base terminal repeat(s) (TR(s)) in cis to generate virus; however, modified AAV TRs and non-AAV TRs including partially or completely synthetic sequences can also serve this purpose. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). The rAAV vector optionally comprises two TRs (e.g., AAV TRs), which generally will be at the 5’ and 3’ ends of the heterologous nucleotide sequence(s), but need not be contiguous thereto. The TRs can be the same or different from each other. The vector genome can also contain a single ITR at its 3’ or 5’ end. The terms “rAAV particle” and “rAAV virion” are used interchangeably here. A “rAAV particle” or “rAAV virion” comprises a rAAV vector genome packaged within an AAV capsid. [0081] The term “terminal repeat” or “TR” includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (ITR) (i.e., mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like). The TR can be an AAV TR or a non-AAV TR. For example, a non-AAV TR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or the SV40 hairpin that serves as the origin of SV40 replication can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and/or addition. Further, the TR can be partially or completely synthetic, such as the “double-D sequence” as described in United States Patent No.5,478,745 to Samulski et al. [0082] Parvovirus genomes have palindromic sequences at both their 5’ and 3’ ends. The palindromic nature of the sequences leads to the formation of a hairpin structure that is stabilized by the formation of hydrogen bonds between the complementary base pairs. This hairpin structure is believed to adopt a “Y” or a “T” shape. See, e.g., FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). [0083] An “AAV terminal repeat” or “AAV TR” may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV now known or later discovered (see, e.g., Table 1). An AAV terminal repeat need not have the native terminal repeat sequence (e.g., a native AAV TR sequence may be altered by insertion, deletion, truncation and/or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and/or provirus rescue, and the like. [0084] Further, the viral capsid or genomic elements can contain other modifications, including insertions, deletions and/or substitutions. [0085] As used herein, parvovirus or AAV “Rep coding sequences” indicate the nucleic acid sequences that encode the parvoviral or AAV non-structural proteins that mediate viral replication and the production of new virus particles. The parvovirus and AAV replication genes and proteins have been described in, e.g., FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). [0086] The “Rep coding sequences” need not encode all of the parvoviral or AAV Rep proteins. For example, with respect to AAV, the Rep coding sequences do not need to encode all four AAV Rep proteins (Rep78, Rep 68, Rep52 and Rep40), in fact, it is believed that AAV5 only expresses the spliced Rep68 and Rep40 proteins. In representative embodiments, the Rep coding sequences encode at least those replication proteins that are necessary for viral genome replication and packaging into new virions. The Rep coding sequences will generally encode at least one large Rep protein (i.e., Rep78/68) and one small Rep protein (i.e., Rep52/40). In particular embodiments, the Rep coding sequences encode the AAV Rep78 protein and the AAV Rep52 and/or Rep40 proteins. In other embodiments, the Rep coding sequences encode the Rep68 and the Rep52 and/or Rep40 proteins. In a still further embodiment, the Rep coding sequences encode the Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52 proteins, or Rep78 and Rep40 proteins. [0087] As used herein, the term “large Rep protein” refers to Rep68 and/or Rep78. Large Rep proteins of the claimed invention may be either wildtype or synthetic. A wildtype large Rep protein may be from any parvovirus or AAV, including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV now known or later discovered (see, e.g., Table 1). A synthetic large Rep protein may be altered by insertion, deletion, truncation and/or missense mutations. [0088] Those skilled in the art will further appreciate that it is not necessary that the replication proteins be encoded by the same polynucleotide. For example, for MVM, the NS-1 and NS-2 proteins (which are splice variants) may be expressed independently of one another. Likewise, for AAV, the p19 promoter may be inactivated and the large Rep protein(s) expressed from one polynucleotide and the small Rep protein(s) expressed from a different polynucleotide. Typically, however, it will be more convenient to express the replication proteins from a single construct. In some systems, the viral promoters (e.g., AAV p19 promoter) may not be recognized by the cell, and it is therefore necessary to express the large and small Rep proteins from separate expression cassettes. In other instances, it may be desirable to express the large Rep and small Rep proteins separately, i.e., under the control of separate transcriptional and/or translational control elements. For example, it may be desirable to control expression of the large Rep proteins, so as to decrease the ratio of large to small Rep proteins. In the case of insect cells, it may be advantageous to down-regulate expression of the large Rep proteins (e.g., Rep78/68) to avoid toxicity to the cells (see, e.g., Urabe et al., (2002) Human Gene Therapy 13:1935). [0089] As used herein, the parvovirus or AAV “cap coding sequences” encode the structural proteins that form a functional parvovirus or AAV capsid (i.e., can package DNA and infect target cells). Typically, the cap coding sequences will encode all of the parvovirus or AAV capsid subunits, but less than all of the capsid subunits may be encoded as long as a functional capsid is produced. Typically, but not necessarily, the cap coding sequences will be present on a single nucleic acid molecule. [0090] The capsid structure of autonomous parvoviruses and AAV are described in more detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott- Raven Publishers). [0091] The extracellular vesicle-targeting zip code sequence may be any nucleotide sequence that increases the amount of mRNA produced from the nucleic acid of interest that is present in EVs and/or increases the percentage of EVs that contain the mRNA. In some embodiments, the extracellular vesicle-targeting zip code sequence, comprises, consists essentially of, or consists of the sequence of SEQ ID NO:1 or a sequence at least 70% identical thereto, e.g., at least 75%, 80%, 85%, 90%, 95%, or 98% identical thereto. [0092] The extracellular vesicle-targeting zip code sequence may be located in any position in the polynucleotide in which it is effective to target the mRNA to EVs. In some embodiments, the extracellular vesicle-targeting zip code sequence is linked to the 3’ end of the polynucleotide. In some embodiments, the polynucleotide is operably linked to a promoter and a poly(A) signal and the extracellular vesicle-targeting zip code sequence is located between the polynucleotide and the poly(A) signal. [0093] In some embodiments, the expression vector is a plasmid that can be used to produce AAV vectors. In some embodiments, the expression vector comprises the sequence of any one of SEQ ID NOS:2, 4, or 6 or a sequence at least 70% identical thereto, e.g., at least 75%, 80%, 85%, 90%, 95%, or 98% identical thereto. [0094] Another aspect of the invention relates to a virus particle comprising the expression vector of the invention. In some embodiments, the virus particle is an AAV particle, an adenovirus particle, a herpesvirus particle, or a baculovirus particle. [0095] In some embodiments, the nucleic acid of interest encodes a protein or nucleic acid. In some embodiments, the protein is an enzyme, a regulatory protein, or a structural protein, e.g., one that can substitute for a missing or defective protein in a subject. The protein may be one that is secreted from a cell or one that is not secreted from a cell. In some embodiments, the nucleic acid is a functional nucleic acid, e.g., an antisense nucleic acid, an inhibitory RNA, or an aptamer. [0096] Any nucleic acid sequence(s) of interest may be delivered in the expression vectors of the present invention. Nucleic acids of interest include nucleic acids encoding polypeptides, including therapeutic (e.g., for medical or veterinary uses), immunogenic (e.g., for vaccines), or diagnostic polypeptides. [0097] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin (including mini- and micro-dystrophins (see, e.g, Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003/017131; International publication WO/2008/088895, Wang et al., Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevic et al., Mol. Ther. 16:657-64 (2008)), myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, clotting factors (e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, the LDL receptor, lipoprotein lipase, ornithine transcarbamylase, ^-globin, ^-globin, spectrin, ^1-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, ^-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase, RP65 protein, cytokines (e.g., ^-interferon, ^-interferon, interferon- ^, interleukin-2, interleukin-4, granulocyte-macrophage colony stimulating factor, lymphotoxin, and the like), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor –3 and –4, brain-derived neurotrophic factor, bone morphogenic proteins [including RANKL and VEGF], glial derived growth factor, transforming growth factor– ^ and – ^, and the like), lysosomal acid ^-glucosidase, ^-galactosidase A, receptors (e.g., the tumor necrosis growth factor ^ soluble receptor), S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effects G-protein coupled receptor kinase type 2 knockdown such as a truncated constitutively active bARKct, anti-inflammatory factors such as IRAP, anti-myostatin proteins, aspartoacylase, and monoclonal antibodies (including single chain monoclonal antibodies; an exemplary Mab is the Herceptin ® Mab). Other illustrative heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptide that has a therapeutic effect in a subject in need thereof. Parvovirus vectors can also be used to deliver monoclonal antibodies and antibody fragments, for example, an antibody or antibody fragment directed against myostatin (see, e.g., Fang et al., Nature Biotechnol.23:584-590 (2005)). [0098] In particular embodiments, the therapeutic protein is heparan alpha-glucosaminide N- acetyltransferase (HGSNAT) or survival motor neuron 1 (SMN1). [0099] Nucleic acid sequences encoding polypeptides include those encoding reporter polypeptides (e.g., an enzyme). Reporter polypeptides are known in the art and include, but are not limited to, Green Fluorescent Protein, ^-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase gene. [0100] Alternatively, in particular embodiments of this invention, the nucleic acid may encode a functional nucleic acid, i.e., nucleic acid that functions without getting translated into a protein, e.g., an antisense nucleic acid, a ribozyme (e.g., as described in U.S. Patent No. 5,877,022), RNAs that effect spliceosome-mediated trans-splicing (see, Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Patent No. 6,013,487; U.S. Patent No. 6,083,702), interfering RNAs (RNAi) including siRNA, shRNA or miRNA that mediate gene silencing (see, Sharp et al., (2000) Science 287:2431), and other non-translated RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Patent No.5,869,248 to Yuan et al.), and the like. Exemplary untranslated RNAs include RNAi against a multiple drug resistance (MDR) gene product (e.g., to treat and/or prevent tumors and/or for administration to the heart to prevent damage by chemotherapy), RNAi against myostatin (e.g., for Duchenne muscular dystrophy), RNAi against VEGF (e.g., to treat and/or prevent tumors), RNAi against phospholamban (e.g., to treat cardiovascular disease, see, e.g., Andino et al., J. Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)); phospholamban inhibitory or dominant-negative molecules such as phospholamban S16E (e.g., to treat cardiovascular disease, see, e.g., Hoshijima et al. Nat. Med. 8:864-871 (2002)), RNAi to adenosine kinase (e.g., for epilepsy), RNAi to a sarcoglycan [e.g., α, β, γ], RNAi against myostatin, myostatin propeptide, follistatin, or activin type II soluble receptor, RNAi against anti-inflammatory polypeptides such as the Ikappa B dominant mutant, and RNAi directed against pathogenic organisms and viruses (e.g., hepatitis B virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.). [0101] Alternatively, in particular embodiments of this invention, the nucleic acid may encode protein phosphatase inhibitor I (I-1), serca2a, zinc finger proteins that regulate the phospholamban gene, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), a molecule that effects G-protein coupled receptor kinase type 2 knockdown such as a truncated constitutively active bARKct; calsarcin, RNAi against phospholamban; phospholamban inhibitory or dominant-negative molecules such as phospholamban S16E, enos, inos, or bone morphogenic proteins (including BNP 2, 7, etc., RANKL and/or VEGF). [0102] The expression vectors may also comprise a nucleic acid that shares homology with and recombines with a locus on a host chromosome. This approach can be utilized, for example, to correct a genetic defect in the host cell. [0103] The present invention also provides expression vectors that express an immunogenic polypeptide, e.g., for vaccination. The nucleic acid may encode any immunogen of interest known in the art including, but not limited to, immunogens from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like. [0104] The use of parvoviruses as vaccine vectors is known in the art (see, e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Patent No. 5,916,563 to Young et al., U.S. Patent No.5,905,040 to Mazzara et al., U.S. Patent No.5,882,652, U.S. Patent No.5,863,541 to Samulski et al.). The antigen may be presented in the parvovirus capsid. Alternatively, the antigen may be expressed from a nucleic acid introduced into a recombinant vector genome. Any immunogen of interest as described herein and/or as is known in the art can be provided by the expression vectors. [0105] An immunogenic polypeptide can be any polypeptide suitable for eliciting an immune response and/or protecting the subject against an infection and/or disease, including, but not limited to, microbial, bacterial, protozoal, parasitic, fungal and/or viral infections and diseases. For example, the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucleoprotein, or an equine influenza virus immunogen) or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein, the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol and env genes products). The immunogenic polypeptide can also be an arenavirus immunogen (e.g., Lassa fever virus immunogen, such as the Lassa fever virus nucleocapsid protein and the Lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as the vaccinia L1 or L8 gene products), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such as NP and GP gene products), a bunyavirus immunogen (e.g., RVFV, CCHF, and/or SFS virus immunogens), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as the human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens) a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and/or any other vaccine immunogen now known in the art or later identified as an immunogen. [0106] Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of the cancer cell. Exemplary cancer and tumor cell antigens are described in S.A. Rosenberg (Immunity 10:281 (1991)). Other illustrative cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, LAGE, NY-ESO- 1, CDK-4, ^-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neu gene product (U.S. Patent No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 proteins, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigens (International Patent Publication No. WO 90/05142); telomerases; nuclear matrix proteins; prostatic acid phosphatase; papilloma virus antigens; and/or antigens now known or later discovered to be associated with the following cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin’s lymphoma, Hodgkin’s lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer and any other cancer or malignant condition now known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med.47:481-91). [0107] It will be understood by those skilled in the art that the nucleic acid(s) of interest can be operably associated with appropriate control sequences. For example, the heterologous nucleic acid can be operably associated with expression control elements, such as transcription/translation control signals, origins of replication, polyadenylation signals (e.g., the soluble neuropilin-1 (sNRP-1) Poly A signal), internal ribosome entry sites (IRES), promoters, and/or enhancers, and the like. [0108] Those skilled in the art will appreciate that a variety of promoter/enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter/enhancer can be constitutive (e.g., the cytomegalovirus promoter or miniature cytomegalovirus promoter) or inducible, depending on the pattern of expression desired. The promoter/enhancer can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. [0109] In particular embodiments, the promoter/enhancer elements can be native to the target cell or subject to be treated. In representative embodiments, the promoters/enhancer element can be native to the nucleic acid sequence. The promoter/enhancer element is generally chosen so that it functions in the target cell(s) of interest. Further, in particular embodiments the promoter/enhancer element is a mammalian promoter/enhancer element. The promoter/enhancer element may be constitutive or inducible. [0110] Inducible expression control elements are typically advantageous in those applications in which it is desirable to provide regulation over expression of the nucleic acid sequence(s). Inducible promoters/enhancer elements for gene delivery can be tissue-specific or –preferred promoter/enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and/or smooth muscle specific or preferred), neural tissue specific or preferred (including brain- specific or preferred), eye specific or preferred (including retina-specific and cornea-specific), liver specific or preferred, bone marrow specific or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoters/enhancer elements include, but are not limited to, a Tet on/off element, a RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter. [0111] In embodiments wherein the nucleic acid sequence(s) is transcribed and then translated in the target cells, specific initiation signals are generally included for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic. Methods of Use [0112] A further aspect of the invention relates to methods of delivering a nucleic acid of interest to bystander cells in a subject, comprising administering to the subject an effective amount of the expression vector, virus particle, or pharmaceutical composition of the invention, thereby forming extracellular vesicles comprising the nucleic acid of interest and delivering the nucleic acid of interest to bystander cells. The method of the invention advantageously may result in a therapeutically significant number of cells expressing the nucleic acid of interest without having to transduce each cell with the expression vector or viral particle. [0113] An additional aspect of the invention relates to methods of treating a disorder treatable by expression of a nucleic acid of interest in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the expression vector, virus particle, or pharmaceutical composition of the invention, thereby treating the disorder. [0114] In some embodiments, the therapeutic protein is HGSNAT and the disorder is mucopolysaccharidosis IIIC. In some embodiments, the therapeutic protein is SMN1 and the disorder is spinal muscle atrophy. In some embodiments, the disorder is fragile X syndrome, Niemann-Pick disease type C, neuronal ceroid lipofuscinoses, Charcot-Marie-Tooth Disease, Friedreich’s Ataxia, or a neuromuscular disorder. [0115] As used herein, “transduction” of a cell by an expression vector, e.g., a virus vector (e.g., an AAV vector) means entry of the vector into the cell and transfer of genetic material into the cell by the incorporation of nucleic acid into the virus vector and subsequent transfer into the cell via the virus vector. [0116] Unless indicated otherwise, “efficient transduction” or “efficient tropism,” or similar terms, can be determined by reference to a suitable positive or negative control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism, respectively, of a positive control or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism, respectively, of a negative control). Similarly, it can be determined if a virus “does not efficiently transduce” or “does not have efficient tropism” for a target tissue, or similar terms, by reference to a suitable control. In particular embodiments, the virus vector does not efficiently transduce (i.e., does not have efficient tropism for) tissues outside the CNS or tissues other than muscle, e.g., liver, kidney, gonads and/or germ cells. In particular embodiments, undesirable transduction of tissue(s) (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of the desired target tissue(s) (e.g., CNS cells or muscle cells). [0117] Those skilled in the art will appreciate that transcription of a heterologous nucleic acid sequence from a viral genome may not be initiated in the absence of trans-acting factors, e.g., for an inducible promoter or otherwise regulated nucleic acid sequence. In the case of a rAAV genome, gene expression from the viral genome may be from a stably integrated provirus and/or from a non-integrated episome, as well as any other form which the virus nucleic acid may take within the cell. [0118] A “bystander cell” is a cell in a subject that has not been transduced by an expression vector after administration of the expression vector to the subject. The ability of the expression vectors of the invention to incorporate mRNA produced from the nucleic acid of interest into EVs, have the EVs deliver the mRNA to bystander cells, and have functional protein expressed in the bystander cells is advantageous in gene therapy methods. [0119] The cell(s) into which the expression vector is introduced can be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons and oligodendrocytes), lung cells, cells of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), blood vessel cells (e.g., endothelial cells, intimal cells), epithelial cells (e.g., gut and respiratory epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells and/or diaphragm muscle cells), dendritic cells, pancreatic cells (including islet cells), hepatic cells, kidney cells, myocardial cells, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and the like. In representative embodiments, the cell can be any progenitor cell. As a further possibility, the cell can be a stem cell (e.g., neural stem cell, liver stem cell). As still a further alternative, the cell can be a cancer or tumor cell. Moreover, the cell can be from any species of origin, as indicated above. Furthermore, the cells may be dividing or non-dividing. [0120] Embodiments of the invention may be performed in vitro or in vivo. One aspect of the present invention is a method of transferring an expression vector to a cell in vitro, e.g., for research purposes or as part of an ex vivo method. The expression vector may be introduced into the cells at the appropriate amount, e.g., multiplicity of infection according to standard transduction methods suitable for the particular target cells. Titers of virus vector to administer can vary, depending upon the target cell type and number, and the particular virus vector, and can be determined by those of skill in the art without undue experimentation. In representative embodiments, at least about 10 3 infectious units, more preferably at least about 10 5 infectious units are introduced to the cell. [0121] In particular embodiments, the cells have been removed from a subject, the expression vector is introduced therein, and the cells are then administered back into the subject. Methods of removing cells from subject for manipulation ex vivo, followed by introduction back into the subject are known in the art (see, e.g., U.S. Patent No.5,399,346). Alternatively, the expression vector can be introduced into cells from a donor subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof (i.e., a “recipient” subject). [0122] Suitable cells for ex vivo gene delivery are as described above. Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 10 8 cells or at least about 10 3 to about 10 6 cells will be administered per dose in a pharmaceutically acceptable carrier. In particular embodiments, the cells transduced with the expression vector are administered to the subject in a treatment effective or prevention effective amount in combination with a pharmaceutical carrier. [0123] The expression vector are additionally useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA. In this manner, the polypeptide or functional RNA can be produced in vivo in the subject. The subject can be in need of the polypeptide because the subject has a deficiency of the polypeptide. Further, the method can be practiced because the production of the polypeptide or functional RNA in the subject may impart some beneficial effect. [0124] The expression vector can also be used to produce a polypeptide of interest or functional RNA in a subject (e.g., using the subject as a bioreactor to produce the polypeptide or to observe the effects of the functional nucleic acid on the subject, for example, in connection with screening methods). The expression vector may also be employed to provide a functional nucleic acid to a cell in vitro or in vivo. Expression of the functional nucleic acid in the cell, for example, can diminish expression of a particular target protein by the cell. Accordingly, functional nucleic acid can be administered to decrease expression of a particular protein in a subject in need thereof. [0125] Expression vector also find use in diagnostic and screening methods, whereby a nucleic acid of interest is transiently or stably expressed in a transgenic animal model. [0126] The expression vector can also be used for various non-therapeutic purposes, including but not limited to use in protocols to assess gene targeting, clearance, transcription, translation, etc., as would be apparent to one skilled in the art. The expression vector can also be used for the purpose of evaluating safety (spread, toxicity, immunogenicity, etc.). Such data, for example, are considered by the United States Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy. Pharmaceutical Formulations, Subjects, and Modes of Administration [0127] Provided according to embodiments of the invention are compositions that include an expression vector or virus particle of the invention. Also provided herein are pharmaceutical compositions comprising an expression vector or virus particle of the invention in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form. The present invention also provides a complex between the expression vector or virus particle in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. By “pharmaceutically acceptable” it is meant a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects. [0128] A further aspect of the invention is a method of administering the expression vector or virus particle of the invention to subjects. Administration of the expression vector or virus particle of the invention to a human subject or an animal in need thereof can be by any means known in the art. Optionally, the expression vector or virus particle of the invention is delivered in a treatment effective or prevention effective dose in a pharmaceutically acceptable carrier. [0129] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one may administer the expression vector or virus particle of the invention in a local rather than systemic manner, for example, in a depot or sustained-release formulation. Further, the expression vector or virus particle of the invention can be delivered adhered to a surgically implantable matrix (e.g., as described in U.S. Patent Publication No. 2004-0013645). The expression vector or virus particle of the invention disclosed herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles comprised of the expression vector or virus particle of the invention, which the subject inhales. The respirable particles can be liquid or solid. Aerosols of liquid particles comprising the expression vector or virus particle of the invention may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No.4,501,729. Aerosols of solid particles comprising the expression vector or virus particle of the invention may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. [0130] In certain embodiments, the expression vector or virus particle of the invention are administered to a subject in need thereof as early as possible in the life of the subject, e.g., as soon as the subject is diagnosed with a disease or disorder. In some embodiments, the method are carried out on a newborn subject, e.g., after newborn screening has identified a disease or disorder. In some embodiments, methods are carried out on a subject prior to the age of 10 years, e.g., prior to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age. In some embodiments, the methods are carried out on juvenile or adult subjects after the age of 10 years. In some embodiments, the methods are carried out on a fetus in utero, e.g., after prenatal screening has identified a disease or disorder. In some embodiments, the methods are carried out on a subject as soon as the subject develops symptoms associated with a disease or disorder. In some embodiments, the methods are carried out on a subject before the subject develops symptoms associated with a disease or disorder, e.g., a subject that is suspected or diagnosed as having a disease or disorder but has not started to exhibit symptoms. [0131] The expression vector or virus particle of the invention may be administered to a subject by any route of administration found to be effective to provide expression of the nucleic acid of interest. The most suitable route will depend on the subject being treated and the disorder or condition being treated. In some embodiments, the expression vector or virus particle of the invention is administered to the subject by a route selected from oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, intravitreal, intracochlear, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intrathecal, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain). [0132] In particular embodiments, more than one administration (e.g., two, three, four or more administrations) may be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, yearly, etc. [0133] The expression vector or virus particle of the invention can be administered to tissues of the CNS (e.g., brain, eye) and may advantageously result in broader distribution of the expression vector or virus particle than would be observed in the absence of the present invention. [0134] Administration can be to any site in a subject, including, without limitation, a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye. [0135] Administration to skeletal muscle according to the present invention includes but is not limited to administration to skeletal muscle in the limbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back, neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/or digits. Suitable skeletal muscles include but are not limited to abductor digiti minimi (in the hand), abductor digiti minimi (in the foot), abductor hallucis, abductor ossis metatarsi quinti, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, articularis genus, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris, depressor labii inferioris, digastric, dorsal interossei (in the hand), dorsal interossei (in the foot), extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi, extensor digitorum, extensor digitorum brevis, extensor digitorum longus, extensor hallucis brevis, extensor hallucis longus, extensor indicis, extensor pollicis brevis, extensor pollicis longus, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimi brevis (in the foot), flexor digitorum brevis, flexor digitorum longus, flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis, iliocostalis lumborum, iliocostalis thoracis, illiacus, inferior gemellus, inferior oblique, inferior rectus, infraspinatus, interspinalis, intertransversi, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator labii superioris, levator labii superioris alaeque nasi, levator palpebrae superioris, levator scapulae, long rotators, longissimus capitis, longissimus cervicis, longissimus thoracis, longus capitis, longus colli, lumbricals (in the hand), lumbricals (in the foot), masseter, medial pterygoid, medial rectus, middle scalene, multifidus, mylohyoid, obliquus capitis inferior, obliquus capitis superior, obturator externus, obturator internus, occipitalis, omohyoid, opponens digiti minimi, opponens pollicis, orbicularis oculi, orbicularis oris, palmar interossei, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis, plantar interossei, plantaris, platysma, popliteus, posterior scalene, pronator quadratus, pronator teres, psoas major, quadratus femoris, quadratus plantae, rectus capitis anterior, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, sartorius, scalenus minimus, semimembranosus, semispinalis capitis, semispinalis cervicis, semispinalis thoracis, semitendinosus, serratus anterior, short rotators, soleus, spinalis capitis, spinalis cervicis, spinalis thoracis, splenius capitis, splenius cervicis, sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius, subscapularis, superior gemellus, superior oblique, superior rectus, supinator, supraspinatus, temporalis, tensor fascia lata, teres major, teres minor, thoracis, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor, and any other suitable skeletal muscle as known in the art. [0136] The expression vector or virus particle of the invention can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion, (optionally, isolated limb perfusion of a leg and/or arm; see, e.g. Arruda et al., (2005) Blood 105: 3458-3464), intrathecal administration, and/or direct intramuscular injection. In particular embodiments, the expression vector or virus particle are administered to a limb (arm and/or leg) of a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra- articular administration. In embodiments of the invention, the expression vector or virus particle can advantageously be administered without employing “hydrodynamic” techniques. Tissue delivery (e.g., to muscle) of prior art vectors is often enhanced by hydrodynamic techniques (e.g., intravenous/intravenous administration in a large volume), which increase pressure in the vasculature and facilitate the ability of the agent to cross the endothelial cell barrier. In particular embodiments, the expression vector or virus particle can be administered in the absence of hydrodynamic techniques such as high volume infusions and/or elevated intravascular pressure (e.g., greater than normal systolic pressure, for example, less than or equal to a 5%, 10%, 15%, 20%, 25% increase in intravascular pressure over normal systolic pressure). Such methods may reduce or avoid the side effects associated with hydrodynamic techniques such as edema, nerve damage and/or compartment syndrome. [0137] Administration to cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle and/or septum. The expression vector or virus particle of the invention can be delivered to cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into left atrium, right atrium, left ventricle, right ventricle), and/or coronary artery perfusion. [0138] Administration to diaphragm muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra-peritoneal administration. [0139] Administration to smooth muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra-peritoneal administration. In one embodiment, administration can be to endothelial cells present in, near, and/or on smooth muscle. [0140] Delivery to a target tissue can also be achieved by delivering a depot comprising the expression vector or virus particle of the invention. In representative embodiments, a depot comprising the expression vector or virus particle is implanted into skeletal, smooth, cardiac and/or diaphragm muscle tissue or the tissue can be contacted with a film or other matrix comprising the expression vector or virus particle. Such implantable matrices or substrates are described in U.S. Patent No.7,201,898. [0141] Administration can also be to a tumor (e.g., in or near a tumor or a lymph node). The most suitable route in any given case will depend on the nature and severity of the condition being treated and/or prevented and on the nature of the particular vector that is being used. [0142] The expression vector or virus particle of the invention may be delivered or targeted to any tissue or organ in the subject. In some embodiments, the expression vector or virus particle are administered to, e.g., a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, the lung, the brain, the spinal cord, the ear, and the eye. In some embodiments, the expression vector or virus particle is administered to a diseased tissue or organ, e.g., a tumor. [0143] In general, the expression vector or virus particle of the invention can be employed to deliver a nucleic acid encoding a polypeptide or functional nucleic acid to treat and/or prevent any disease state for which it is beneficial to deliver a therapeutic polypeptide or functional nucleic acid. Illustrative disease states include, but are not limited to: fragile X syndrome, neuronal ceroid lipofuscinoses, Charcot-Marie-Tooth Disease, Friedreich’s Ataxia, cystic fibrosis (cystic fibrosis transmembrane regulator protein) and other diseases of the lung, hemophilia A (Factor VIII), hemophilia B (Factor IX), thalassemia (ß-globin), anemia (erythropoietin) and other blood disorders, Alzheimer’s disease (GDF; neprilysin), multiple sclerosis (ß-interferon), Parkinson’s disease (glial-cell line derived neurotrophic factor [GDNF]), Huntington’s disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAi including RNAi against VEGF or the multiple drug resistance gene product), diabetes mellitus (insulin), muscular dystrophies including Duchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, a sarcoglycan [e.g., α, β, γ], RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin, mini-utrophin, RNAi against splice junctions in the dystrophin gene to induce exon skipping [see, e.g., WO/2003/095647], antisense against U7 snRNAs to induce exon skipping [see, e.g., WO/2006/021724], and antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker, Gaucher disease (glucocerebrosidase), Hurler’s disease (α-L- iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic defects, congenital emphysema (α1-antitrypsin), Lesch-Nyhan Syndrome (hypoxanthine guanine phosphoribosyl transferase), Niemann-Pick disease (sphingomyelinase), Tays Sachs disease (lysosomal hexosaminidase A), Maple Syrup Urine Disease (branched-chain keto acid dehydrogenase), retinal degenerative diseases (and other diseases of the eye and retina; e.g., PDGF for macular degeneration), diseases of solid organs such as brain (including Parkinson’s Disease [GDNF], astrocytomas [endostatin, angiostatin and/or RNAi against VEGF], glioblastomas [endostatin, angiostatin and/or RNAi against VEGF]), liver, kidney, heart including congestive heart failure or peripheral artery disease (PAD) (e.g., by delivering protein phosphatase inhibitor I (I-1), serca2a, zinc finger proteins that regulate the phospholamban gene, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effects G- protein coupled receptor kinase type 2 knockdown such as a truncated constitutively active bARKct; calsarcin, RNAi against phospholamban; phospholamban inhibitory or dominant- negative molecules such as phospholamban S16E, etc.), arthritis (insulin-like growth factors), joint disorders (insulin-like growth factor 1 and/or 2), intimal hyperplasia (e.g., by delivering enos, inos), improve survival of heart transplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNFα soluble receptor), hepatitis ( ^-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe’s disease (galactocerebrosidase), Batten’s disease, spinal cerebral ataxias including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), neuromuscular disorders, autoimmune diseases, and the like. The invention can further be used following organ transplantation to increase the success of the transplant and/or to reduce the negative side effects of organ transplantation or adjunct therapies (e.g., by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production). As another example, bone morphogenic proteins (including BNP 2, 7, etc., RANKL and/or VEGF) can be administered with a bone allograft, for example, following a break or surgical removal in a cancer patient. [0144] In particular embodiments, expression vector or virus particle of the invention is administered to skeletal muscle, diaphragm muscle and/or cardiac muscle (e.g., to treat and/or prevent muscular dystrophy or heart disease [for example, PAD or congestive heart failure]). [0145] Gene transfer has substantial potential use for understanding and providing therapy for disease states. There are a number of inherited diseases in which defective genes are known and have been cloned (i.e., disorders treatable by expression of a nucleic acid of interest). In general, the above disease states fall into two classes: deficiency states, usually of proteins/enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically inherited in a recessive manner. For deficiency state diseases, gene transfer can be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations. For unbalanced disease states, gene transfer can be used to create a disease state in a model system, which can then be used in efforts to counteract the disease state. Thus, expression vectors permit the treatment and/or prevention of genetic diseases. [0146] As a further aspect, the expression vectors of the present invention may be used to produce an immune response in a subject. According to this embodiment, expression vectors comprising a nucleic acid sequence encoding an immunogenic polypeptide can be administered to a subject, and an active immune response is mounted by the subject against the immunogenic polypeptide. Immunogenic polypeptides are as described hereinabove. In some embodiments, a protective immune response is elicited. [0147] Alternatively, the expression vectors may be administered to a cell ex vivo and the altered cell is administered to the subject. The expression vectors comprising the nucleic acid is introduced into the cell, and the cell is administered to the subject, where the nucleic acid encoding the immunogen can be expressed and induce an immune response in the subject against the immunogen. In particular embodiments, the cell is an antigen-presenting cell (e.g., a dendritic cell). [0148] An “active immune response” or “active immunity” is characterized by “participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both.” Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to an immunogen by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the “transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host.” Id. [0149] A “protective” immune response or “protective” immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence of disease. Alternatively, a protective immune response or protective immunity may be useful in the treatment and/or prevention of disease, in particular cancer or tumors (e.g., by preventing cancer or tumor formation, by causing regression of a cancer or tumor and/or by preventing metastasis and/or by preventing growth of metastatic nodules). The protective effects may be complete or partial, as long as the benefits of the treatment outweigh any disadvantages thereof. In particular embodiments, the expression vector or cell comprising the nucleic acid can be administered in an immunogenically effective amount, as described below. [0150] The expression vectors can also be administered for cancer immunotherapy by administration of expression vectors expressing one or more cancer cell antigens (or an immunologically similar molecule) or any other immunogen that produces an immune response against a cancer cell. To illustrate, an immune response can be produced against a cancer cell antigen in a subject by administering expression vectors comprising a nucleic acid encoding the cancer cell antigen, for example to treat a patient with cancer and/or to prevent cancer from developing in the subject. The expression vectors may be administered to a subject in vivo or by using ex vivo methods, as described herein. Alternatively, the cancer antigen can be expressed as part of the expression vectors. [0151] As another alternative, any other therapeutic nucleic acid (e.g., RNAi) or polypeptide (e.g., cytokine) known in the art can be administered to treat and/or prevent cancer. [0152] As used herein, the term “cancer” encompasses tumor-forming cancers. Likewise, the term “cancerous tissue” encompasses tumors. A “cancer cell antigen” encompasses tumor antigens. [0153] The term “cancer” has its understood meaning in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize). Exemplary cancers include, but are not limited to melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin’s lymphoma, Hodgkin’s lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer and any other cancer or malignant condition now known or later identified. In representative embodiments, the invention provides a method of treating and/or preventing tumor-forming cancers. [0154] The term “tumor” is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used to prevent and treat malignant tumors. [0155] By the terms “treating cancer,” “treatment of cancer” and equivalent terms it is intended that the severity of the cancer is reduced or at least partially eliminated and/or the progression of the disease is slowed and/or controlled and/or the disease is stabilized. In particular embodiments, these terms indicate that metastasis of the cancer is prevented or reduced or at least partially eliminated and/or that growth of metastatic nodules is prevented or reduced or at least partially eliminated. [0156] By the terms “prevention of cancer” or “preventing cancer” and equivalent terms it is intended that the methods at least partially eliminate or reduce and/or delay the incidence and/or severity of the onset of cancer. Alternatively stated, the onset of cancer in the subject may be reduced in likelihood or probability and/or delayed. [0157] In particular embodiments, cells may be removed from a subject with cancer and contacted with the expression vector or virus particle of the invention. The modified cell is then administered to the subject, whereby an immune response against the cancer cell antigen is elicited. This method can be advantageously employed with immunocompromised subjects that cannot mount a sufficient immune response in vivo (i.e., cannot produce enhancing antibodies in sufficient quantities). [0158] It is known in the art that immune responses may be enhanced by immunomodulatory cytokines (e.g., ^-interferon, ^-interferon, ^-interferon, ^-interferon, ^-interferon, interleukin-1 ^, interleukin-1 ^, interleukin-2, interleukin-3, interleukin-4, interleukin 5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin 12, interleukin-13, interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumor necrosis factor- ^, tumor necrosis factor- ^, monocyte chemoattractant protein-1, granulocyte- macrophage colony stimulating factor, and lymphotoxin). Accordingly, immunomodulatory cytokines (preferably, CTL inductive cytokines) may be administered to a subject in conjunction with the expression vector or virus particle. [0159] Cytokines may be administered by any method known in the art. Exogenous cytokines may be administered to the subject, or alternatively, a nucleic acid encoding a cytokine may be delivered to the subject using a suitable vector, and the cytokine produced in vivo. [0160] The methods of the present invention find use in both veterinary and medical applications. Suitable subjects include avians, reptiles, amphibians, fish, and mammals. The term “mammal” as used herein includes, but is not limited to, humans, primates, non-human primates (e.g., monkeys and baboons), cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats, mice, hamsters, and the like), etc. Human subjects include neonates, infants, juveniles, and adults. Optionally, the subject is “in need of” the methods of the present invention, e.g., because the subject has or is believed at risk for a disorder including those described herein or that would benefit from the delivery of a polynucleotide including those described herein. As a further option, the subject can be a laboratory animal and/or an animal model of disease. Preferably, the subject is a human. [0161] In some embodiments, the expression vector or virus particle of the invention is introduced into a cell and the cell can be administered to a subject to elicit an immunogenic response against the delivered polypeptide (e.g., expressed as a transgene or in the capsid). Typically, a quantity of cells expressing an immunogenically effective amount of the polypeptide in combination with a pharmaceutically acceptable carrier is administered. An “immunogenically effective amount” is an amount of the expressed polypeptide that is sufficient to evoke an active immune response against the polypeptide in the subject to which the pharmaceutical formulation is administered. In particular embodiments, the dosage is sufficient to produce a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof. [0162] The expression vector or virus particle of the invention can further be administered to elicit an immunogenic response (e.g., as a vaccine). Typically, immunogenic compositions of the present invention comprise an immunogenically effective amount of the expression vector or virus particle in combination with a pharmaceutically acceptable carrier. Optionally, the dosage is sufficient to produce a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof. Subjects and immunogens are as described above. [0163] Dosages of the expression vector (e.g., viral vector) to be administered to a subject depend upon the mode of administration, the disease or condition to be treated and/or prevented, the individual subject’s condition, the particular expression vector, and the nucleic acid to be delivered, and the like, and can be determined in a routine manner. Exemplary doses for achieving therapeutic effects are titers of at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 transducing units, optionally about 10 8 – 10 15 transducing units. In some embodiments, the dosage of the expression vector is less than what would be needed to achieve a therapeutic effect with an expression vector that does not comprise an extracellular vesicle-targeting zip code sequence. [0164] In a representative embodiment, the invention provides a method of treating and/or preventing muscular dystrophy in a subject in need thereof, the method comprising: administering a treatment or prevention effective amount of expression vector or virus particle of the invention to a mammalian subject, wherein the expression vector or virus particle comprises a nucleic acid encoding dystrophin, a mini-dystrophin, a micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin, a micro-dystrophin, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, an antibody or antibody fragment against myostatin or myostatin propeptide, and/or RNAi against myostatin. In particular embodiments, the expression vector or virus particle can be administered to skeletal, diaphragm and/or cardiac muscle as described elsewhere herein. [0165] Alternatively, the invention can be practiced to deliver a nucleic acid to skeletal, cardiac or diaphragm muscle, which is used as a platform for production of a polypeptide (e.g., an enzyme) or functional nuclei acid (e.g., functional RNA, e.g., RNAi, microRNA, antisense RNA) that normally circulates in the blood or for systemic delivery to other tissues to treat and/or prevent a disorder (e.g., a metabolic disorder, such as diabetes (e.g., insulin), hemophilia (e.g., Factor IX or Factor VIII), a mucopolysaccharide disorder (e.g., Sly syndrome, Hurler Syndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter’s Syndrome, Sanfilippo Syndrome A, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.) or a lysosomal storage disorder (such as Gaucher’s disease [glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] or Fabry disease [ ^-galactosidase A]) or a glycogen storage disorder (such as Pompe disease [lysosomal acid ^ glucosidase]). Other suitable proteins for treating and/or preventing metabolic disorders are described above. The use of muscle as a platform to express a nucleic acid of interest is described in U.S. Patent Publication No.2002/0192189. [0166] Thus, as one aspect, the invention further encompasses a method of treating and/or preventing a metabolic disorder in a subject in need thereof, the method comprising: administering a treatment or prevention effective amount of expression vector or virus particle of the invention to a subject (e.g., to skeletal muscle of a subject), wherein the expression vector or virus particle comprises a nucleic acid encoding a polypeptide, wherein the metabolic disorder is a result of a deficiency and/or defect in the polypeptide. Illustrative metabolic disorders and nucleic acids encoding polypeptides are described herein. Optionally, the polypeptide is secreted (e.g., a polypeptide that is a secreted polypeptide in its native state or that has been engineered to be secreted, for example, by operable association with a secretory signal sequence as is known in the art). Without being limited by any particular theory of the invention, according to this embodiment, administration to the skeletal muscle can result in secretion of the polypeptide into the systemic circulation and delivery to target tissue(s). Methods of delivering the expression vector or virus particle of the invention to skeletal muscle are described in more detail herein. [0167] The invention can also be practiced to produce antisense RNA, RNAi or other functional RNA (e.g., a ribozyme) for systemic delivery. [0168] The invention also provides a method of treating and/or preventing congenital heart failure or PAD in a subject in need thereof, the method comprising administering a treatment or prevention effective amount of the expression vector or virus particle of the invention to a mammalian subject, wherein the expression vector or virus particle comprises a nucleic acid encoding, for example, a sarcoplasmic endoreticulum Ca 2+ -ATPase (SERCA2a), an angiogenic factor, phosphatase inhibitor I (I-1), RNAi against phospholamban; a phospholamban inhibitory or dominant-negative molecule such as phospholamban S16E, a zinc finger protein that regulates the phospholamban gene, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, a β-adrenergic receptor kinase inhibitor (βARKct), inhibitor 1 of protein phosphatase 1, S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effects G-protein coupled receptor kinase type 2 knockdown such as a truncated constitutively active bARKct, Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir- 206 and/or mir-208. [0169] In particular embodiments, the expression vector or virus particle of the invention may be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders and tumors. Illustrative diseases of the CNS include, but are not limited to Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Canavan disease, Leigh’s disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, spinal muscle atrophy, progressive muscular atrophy, mucopolysaccharidosis, fragile X syndrome, neuronal ceroid lipofuscinoses, Charcot-Marie-Tooth Disease, Friedreich’s Ataxia, Pick’s disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger’s disease, trauma due to spinal cord or head injury, Tay Sachs disease, Lesch-Nyan disease, epilepsy, cerebral infarcts, psychiatric disorders including mood disorders (e.g., depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder), schizophrenia, drug dependency (e.g., alcoholism and other substance dependencies), neuroses (e.g., anxiety, obsessional disorder, somatoform disorder, dissociative disorder, grief, post-partum depression), psychosis (e.g., hallucinations and delusions), dementia, paranoia, attention deficit disorder, psychosexual disorders, sleeping disorders, pain disorders, eating or weight disorders (e.g., obesity, cachexia, anorexia nervosa, and bulemia) and cancers and tumors (e.g., pituitary tumors) of the CNS. [0170] Disorders of the CNS include ophthalmic disorders involving the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma). [0171] Most, if not all, ophthalmic diseases and disorders are associated with one or more of three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. The expression vector or virus particle of the invention can be employed to deliver anti-angiogenic factors; anti-inflammatory factors; factors that retard cell degeneration, promote cell sparing, or promote cell growth and combinations of the foregoing. [0172] Diabetic retinopathy, for example, is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors either intraocularly (e.g., in the vitreous) or periocularly( e.g., in the sub-Tenon’s region). One or more neurotrophic factors may also be co-delivered, either intraocularly (e.g., intravitreally) or periocularly. [0173] Uveitis involves inflammation. One or more anti-inflammatory factors can be administered by intraocular (e.g., vitreous or anterior chamber) administration of a expression vector or virus particle of the invention. [0174] Retinitis pigmentosa, by comparison, is characterized by retinal degeneration. In representative embodiments, retinitis pigmentosa can be treated by intraocular (e.g., vitreal administration) of the expression vector or virus particle of the invention encoding one or more neurotrophic factors. [0175] Age-related macular degeneration involves both angiogenesis and retinal degeneration. This disorder can be treated by administering the expression vector or virus particle of the invention encoding one or more neurotrophic factors intraocularly (e.g., vitreous) and/or one or more anti-angiogenic factors intraocularly or periocularly (e.g., in the sub-Tenon’s region). [0176] Glaucoma is characterized by increased ocular pressure and loss of retinal ganglion cells. Treatments for glaucoma include administration of one or more neuroprotective agents that protect cells from excitotoxic damage using the expression vector or virus particle of the invention. Such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors, delivered intraocularly, optionally intravitreally. [0177] other embodiments, the present invention may be used to treat seizures, e.g., to reduce the onset, incidence or severity of seizures. The efficacy of a therapeutic treatment for seizures can be assessed by behavioral (e.g., shaking, ticks of the eye or mouth) and/or electrographic means (most seizures have signature electrographic abnormalities). Thus, the invention can also be used to treat epilepsy, which is marked by multiple seizures over time. [0178] In one representative embodiment, somatostatin (or an active fragment thereof) is administered to the brain using the expression vector or virus particle of the invention to treat a pituitary tumor. According to this embodiment, the expression vector or virus particle encoding somatostatin (or an active fragment thereof) is administered by microinfusion into the pituitary. Likewise, such treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary). The nucleic acid (e.g., GenBank Accession No. J00306) and amino acid (e.g., GenBank Accession No. P01166; contains processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatins as are known in the art. [0179] In particular embodiments, the expression vector or virus particle of the invention can comprise a secretory signal as described in U.S. Patent No.7,071,172. [0180] In representative embodiments of the invention, the expression vector or virus particle of the invention is administered to the CNS (e.g., to the brain or to the eye). The expression vector or virus particle may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and portaamygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus. The expression vector or virus particle may also be administered to different regions of the eye such as the retina, cornea and/or optic nerve. [0181] The expression vector or virus particle of the invention may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the expression vector or virus particle. The expression vector or virus particle may further be administered intravascularly to the CNS in situations in which the blood-brain barrier has been perturbed (e.g., brain tumor or cerebral infarct). [0182] The expression vector or virus particle of the invention can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon’s region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons. [0183] In particular embodiments, the expression vector or virus particle of the invention is administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired region or compartment in the CNS. In other embodiments, the expression vector or virus particle may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye, may be by topical application of liquid droplets. As a further alternative, the expression vector or virus particle may be administered as a solid, slow-release formulation (see, e.g., U.S. Patent No.7,201,898). [0184] In yet additional embodiments, the expression vector or virus particle of the invention can used for retrograde transport to treat and/or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.). For example, the expression vector or virus particle can be delivered to muscle tissue from which it can migrate into neurons. [0185] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.
EXAMPLES Example 1 MPS IIIC Studies [0186] In preliminary studies, the disclosed rAAV product was constructed by incorporation of an EV-mRNA packaging signal to a rAAV-hHGSNAT gene replacement vector to facilitate the by-stander effect. The vector construct was tested using an in vitro model of MPS IIIC, to assess its therapeutic potential. [0187] Construction of rAAV-hHGSNAT vectors: A rAAV vector plasmid, ptr-CMV- hHGSNAT zc (SEQ ID NO:2), was constructed to express hHGSNAT, containing a 25 nt EV zip-code (ZC) signal sequence for packaging hHGSNAT-mRNA into EVs. A second AAV vector plasmid, ptr-CMV-hHGSNAT (SEQ ID NO:3), without the EV signal was constructed as a control. These plasmids were used to produce rAAV-CMV-hHGSNAT zc and rAAV-CMV- hHGSNAT viral vectors. The rAAV-hHGSNAT viral vector genome (vg) contains only minimal elements required for transgene expression (FIG.1). [0188] rAAV-mediated expression of functional rHGSNAT and EV-hHGSNAT-mRNA packaging: To determine the function of these vector constructs, they were first tested in vitro in Hela cells by transfection with the vector plasmids, ptr-CMV-hHGSNAT or ptr-CMV- hHGSNAT zc . At 48 h post transfection, cell lysates were assayed for rHGSNAT expression by HGSNAT activity assay and qRT-PCR. The results showed significantly increased HGSNAT activity and mRNA in the transfected cells, compared to non-transfected Hela cells These data indicate both vectors mediated effective expression of functional rHGSNAT. [0189] To further assess the therapeutic potential of the rAAV-hHGSNAT vectors, they were tested in human MPS IIIC skin fibroblast (GM05157) cultures by transfection with the vector plasmids and by infection with rAAV2 viral vectors. Healthy human skin fibroblasts (GM00969) and untreated GM05157 cells were used as controls. GM05157 cells in 10 cm plates were transfected with plasmid ptr-CMV-hHGSNAT or ptr-CMV-hHGSNAT zc (7 μg/plate), or infected with rAAV2-CMV-hHGSNAT or rAAV2-CMV-hHGSNAT zc viral vectors (1x10 3 vg/cell). At 48 h post transduction, cell lysates were assayed in duplicate for rHGSNAT expression by HGSNAT activity assay and qRT-PCR (FIGS. 2A-2B). Media samples were processed for EV extraction, transfer, and analysis. As a result, both AAV vector constructs mediated effective rHGSNAT expression by transfection (FIG. 2A) and rAAV2 infection (FIG 2B). While the mechanisms are unclear, inclusion of the 25 nt EV ZC signal sequence was shown to enhance rHGSNAT expression at both the protein and mRNA levels. Transfections appear to induce higher levels of rHGSNAT expression than infection, likely because more vector plasmids could enter the cells (via bulk transport) than rAAV2 viral vectors (via receptor- mediated uptake). Notably, the experiments of transfection and vector infection were conducted in duplicate and repeated ≥2 times. [0190] To determine whether including the 25 nt EV ZC signal sequence in the vector mediates EV-HGSNAT-mRNA packaging, media samples from the transduced and control cells were processed to isolate EVs by ultracentrifugation. The isolated EVs were processed to extract RNA and EV RNA samples were assayed for hHGSNAT mRNA by qRT-PCR. The results showed that both AAV-hHGSNAT zc and AAV-hHGSNAT vectors mediated the secretion of hHGSNAT-mRNA-containing EVs from cells transduced by transfection (FIG. 3A) and infection (FIG.3B). However, the EV ZC tag was shown to mediate much higher levels of EV- hHGSNAT-mRNA packaging. While the mechanism is unclear, it is possible that the transduction of AAV-hHGSNAT vectors without EV ZC may also induce EV-hHGSNAT- mRNA packaging as a biophysiological response. Similarly, plasmid transfections appear to induce much higher levels of EV rHGSNAT mRNA than rAAV2 infection, congruent with the previously observed higher level intracellular expression. Together, these observations support the hypothesis that the EV ZC signal can mediate effective EV-hHGSNAT-mRNA cargo by AAV gene delivery. [0191] Characteristics of AAV-mediated EVs in human MPS IIIC skin fibroblasts: The EV samples were characterized using NanoSight NS500 at UNC Nanomedicines Characterization Core Facility, to visualize the EVs, and determine the quantity and size distribution of EVs. The isolated EVs were shown to be medium sized (111.6–144.8 nm) (FIGS. 4A-4B). Interestingly, while the same number of cells were used among different experimental groups, about 2-fold more EVs were detected from non-treated MPS IIIC cells than from healthy cells or MPS IIIC cells treated by transfection (FIG. 4A) or infection (FIG. 4B) with both of the AAV-hHGSNAT vector constructs with or without the EV ZC signal. Notably, an increase in EVs has been linked to many pathological conditions, such as autoimmune diseases and cancer. Therefore, the increase in EVs from MPS IIIC cells may be due to the disease pathophysiology. Importantly, the EV reduction following AAV-hHGSNAT transduction suggests the correction of the pathological condition, supporting the therapeutic potential of AAV-hHGSNAT gene therapy. [0192] EV-mRNA transfer leading to the expression of functional rHGSNAT protein in recipient MPS IIIC cells: To determine whether AAV-hHGSNAT-mediated EVs can facilitate bystander effects, GM05157 cells were incubated in duplicate with EVs that had been isolated from the media of MPS IIIC cells treated with AAV-hHGSNAT vectors by plasmid transfection (FIG. 5A) or AAV2 infection (FIG. 5B). At 48 h of incubation, the recipient cell lysates were assayed for HGSNAT enzyme activity. The results showed that EVs from both transfected or infected cells resulted in HGSNAT activity in MPS IIIC cells, indicating that HGSNAT-mRNA was transferred to the cells by EVs and translated into functional HGSNAT protein. EVs from cells transduced with AAV-hHGSNAT zc led to higher mRNA transfer (FIGS. 2A-2B and 3A- 3B). Again, transfection appears to induce more EV-mediated rHGSNAT protein expression (FIGS. 5A-5B), compared to rAAV2 infection. These data demonstrate that tagging the 25 nt EV ZC signal to rAAV-hHGSNAT vector facilitates potentially efficacious bystander effects in untransduced MPS IIIC cells, supporting the therapeutic potential of this approach for treating neurogenetic diseases involving non-secreted proteins. [0193] Clearance of GAG storage in vitro in MPS IIIC cells by rAAV-hHGSNAT gene transfer and EV-hHGSNAT-mRNA cargo: To further assess the function of the rAAV9- mediated rHGSNAT, human MPS IIIC skin fibroblasts (GM05157) were transfected in duplicate with plasmid ptr-CMV-hHGSNAT or ptr-CMV-hHGSNAT zc (7 μg/plate), and the media from transfected cells were used to culture non-treated GM05157 cells. At 48 h post transfection or media incubation, cell lysates were assayed in duplicate for GAG content. The results showed the reduction of GAG content to normal levels in cells transduced with both vector constructs (FIG. 6A), further demonstrating that the AAV-mediated rHGSNAT are functional and led to the clearance of GAG storage in MPS IIIC cells. Importantly, incubation with media of hHGSNAT zc -transduced cells resulted in complete correction of GAG storage in MPS IIIC cells (FIG. 6B), indicating that ZC-mediated EV cargo led to the translation of HGSNAT-mRNA to functional rHGSNAT protein in non-transduced MPS IIIC cells. Notably, incubation with media from hHGSNAT-transduced cells led to partial GAG reduction in MPS IIIC cells (FIG. 6B), suggesting that transduction by ptr-hHGSNAT vector may also result in a low level of EV- HGSNAT-mRNA packaging, though the mechanisms are unclear. Correlating with data presented in FIGS.2A-2B, 3A-3B, and 5A-5B, these results further demonstrate the therapeutic potential of the bystander effects facilitated by the AAV-mediated EV-hHGSNAT-mRNA cargo for treating MPS IIIC. The EV-facilitated cross-correction allows significant therapeutic benefit from a small number of transduced cells within a tissue, as long as they are well distributed. [0194] In summary, an rAAV-hHGSNAT vector containing a 25 nt EV ZC for mRNA cargo was developed. The studies showed that the rAAV-hHGSNAT zc mediates efficient rHGSNAT expression and EV-HGSNAT-mRNA packaging. Importantly, these EVs can transfer HGSNAT-mRNA to and mediate the expression of rHGSNAT protein in untreated MPS IIIC cells. These data demonstrate that rAAV-hHGSNAT vector containing the 25 nt EV ZC can mediate not only efficient rHGSNAT expression, but also EV-HGSNAT-mRNA cargo. The EVs can transfer their rHGSNAT-mRNA contents to the recipient cells, where the rHGSNAT- mRNA can be translated to functional rHGSNAT protein leading to the clearance of GAG storage. The demonstrated EV-facilitated bystander effect offers great potential for treating MPS IIIC using rAAV gene replacement therapy. This bystander effect may allow optimal therapeutic benefits via cross-correction, without having to transduce every single cell, because the transduced cells serve as factories producing rHGSNAT-mRNA-containing EVs that can enter the neighboring cells, where the rHGSNAT-mRNAs translate to functional proteins. [0195] AAV normally packages a single-stranded (ss) DNA genome that must be converted to double-strand (ds) DNA after infection. Constructs less than half or less of the normal WT AAV genome size can be packaged as dimeric inverted repeat DNA molecules. These self- complementary (sc) genomes fold into ds DNA as soon as they are released from the capsid, bypassing the requirement for DNA synthesis by the host cells to convert the single-stranded (ss) vg into active double-stranded DNA and providing faster and more efficient transduction. [0196] A set of two self-complementary AAV (sc) vector expressing rhHGSNAT were constructed for added transduction efficiency. The scAAV-mCMV-hHGSNAT zc vector (SEQ ID NO:4) was constructed that contains the 25 nt ZC sequence linked to hHGSNAT cDNA and the scAAV-mCMV-hHGSNAT vector (SEQ ID NO:5) without the ZC signal. To accommodate the vector genome size limit for AAV vector packaging (~4,700 bp), a 228 bp truncated miniature CMV promoter (mCMV), and a 16 bp soluble neuropilin-1 (sNRP-1) Poly A signal were used in these constructs, resulting in the 4,75 0bp and 4,812 bp vector genome respectively (FIG. 7). These plasmids are used to produce scAAV-mCMV-hHGSNAT zc and scAAV-mCMV- hHGSNAT viral vectors. [0197] To determine whether the ptrsk-mCMV-hHGSNAT vector constructs generate scAAV vector, 3-plasmid-cotransfection was performed in HEK293 cells to produce scAAV9-CMV- hHGSNAT ZC and scAAV9-CMV-hHGSNAT. As controls, pTR-CMV-hHGSNAT constructs were used to produce ssAAV-hHGSNAT vectors. The purified vector products were analyzed by alkaline denaturing gel electrophoresis. The results showed that the AAV-mCMV- hHGSNAT ZC and AAV-mCMV-hHGSNAT viral vector products were scAAV vectors (Fig. 8). In contrast, the control AAV-CMV-hHGSNAT ZC and AAV-CMV-hHGSNAT viral vector products were single-stranded AAV vectors, based on the mobility of the viral genome on the alkaline denaturing gel. These results indicate that using small mCMV promotor and SNRP-1 Poly A signal enabled the vg being packaged into scAAV-mCMV-hHGSNAT ZC and scAAV- mCMV-hHGSNAT ZC vectors. Example 2 SMA Studies [0198] The disclosed rAAV product was constructed by incorporation of EV-mRNA packaging signal to a scAAV-hSMN1 gene replacement vector to facilitate the bystander effect. The vector construct was tested using an in vitro model of MPS IIIC, to assess its therapeutic potential. [0199] Construction of rAAV-hSMN1 vectors: A rAAV vector plasmid, ptrsk-CMV- hSMN1 zc (SEQ ID NO:6), was constructed to express hSMN protein, containing a 25 nt EV zip- code (ZC) signal sequence for packaging hSMN1-mRNA into EVs. A second AAV vector plasmid, ptrsk-CMV-hSMN1 (SEQ ID NO:7), without the EV signal, was constructed as a control. These plasmids are used to produce scAAV-CMV-hSMN1 zc and scAAV-CMV-hSMN1 viral vectors. The rAAV-hSMN1 viral vector genome (vg) contains only minimal elements required for transgene expression (FIG.9). [0200] rAAV-mediated expression of rSMN and functional EV-hSMN1-mRNA cargo in vitro in human SMN1 skin fibroblasts: To determine the function of these scAAV-hSMN1 vector constructs, human SMN1 skin fibroblasts (GM00232) were transfected with the vector plasmids (FIGS.10A-10B), or incubated with EVs extracted from the vector plasmid transfected cells (FIGS.10C-10D). At 48 h post transfection or 24 h post EV incubation, cell samples were assayed for SMN expression by western blot and qRT-PCR. Higher levels of SMN1 protein and SMN1 mRNA were detected in cells transfected with both vector plasmids (FIGS. 10A-10B). Increases in SMN1 protein and SMN1 mRNA were also detected in cells that were incubated with the EVs extracted from the media of hSMN1 zc -transfected cells (FIGS. 10C-10D). These data demonstrate that both the hSMN1 zc and hSMN1 vectors mediated efficient rSMN expression, and importantly, EV-SMN1-mRNA cargo, leading to the translation of SMN1- mRNA to rSMN protein in recipient SMA cells. These data support the hypothesis on the potential of EV-facilitated bystander effect that may lead to the cross-correction of SMN defects in non-transduced cells. This bystander effect will allow the maximal therapeutic benefits from a relatively small number of transduced cells within a tissue, as long as they are well distributed, without having to transduce 100% of the cells. It may therefore significantly reduce the vector dose needed for optimal therapeutic efficacy for treating SMN in humans, and ease one of the critical challenges in translation, the scale-up manufacturing. [0201] In conclusion, the novel AAV-SMN1 vector with the 25 nt EV ZC will mediate the bystander effect of rSMN1 via EV-rSMN1-mRNA cargo and significantly improve the efficacy of AAV gene replacement therapy for treating SMA. This rAAV mediated EV-mRNA cargo approach is applicable for gene replacement therapy treating many neurogenetic diseases involving a non-secreted protein. References 1. Andrade, F., Aldamiz-Echevarria, L., Llarena, M. & Couce, M.L. Sanfilippo syndrome: Overall review. Pediatr Int 57, 331-8 (2015). 2. Lavery, C., Hendriksz, C.J. & Jones, S.A. Mortality in patients with Sanfilippo syndrome. Orphanet J Rare Dis 12, 168 (2017). 3. Foust, K.D. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27, 59-65 (2009). 4. Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 16, 1073-80 (2008). 5. Duque, S. et al. 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Proc Natl Acad Sci U S A 114, E8987-E8995 (2017). 65. Yanshina, D.D. et al. Structural features of the interaction of the 3'-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting. Biochimie 144, 134-143 (2018). [0202] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims. Sequences Extracellular vesicle-targeting zip code sequence (SEQ ID NO:1) ACCCTGCCGCCTGGACTCCGCCTGT pTR-CMV-hHGSNAT zc (SEQ ID NO:2) GGGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACC AAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG AGAGGGA GTGGCCAACTCCATCACTAGGGGTTCCTAGATCTGAATTCGGTACCCGTTACATAACTTA CGGTAAATG GCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTC CCATAGTA ACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCAC TTGGCAGT ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC CGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTA TTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTT CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGAC TTTCCAAA ATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGT CTATATAA GCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACC TCCATAGA AGACACCGGGACCGATCCAGCCTCCGGACTCTAGAATGAGCGGGGCGGGCAGGGCGCTGG CCGCGCT GCTGCTGGCCGCGTCCGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCG CGATGCCC AGGCCGCGCCGCCACGAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGG CTTTGCT ACTCATCCATAATGAACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTG TTATCACTG CTTGTTTCAGGTTCTGGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGC AGCTGCCT CTGTCAGCACCCAGCACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAG TTTGTAGG TTGGAATACAGATTTGGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAAT GGAGTTAG TGAAATTGCCTGTGACCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAG CATTGCATT CCTTATTGGTCTTGCTGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGA TGACTTTAAC AATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTG GGATCTCCC AGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTG CCGCCCCG CCTCCGCAGCGTGGACACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTCAATTA TGGAGGAGG AAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTT CCCGTGGT TTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGT GTTCAAAATT CAGATTGCTGGGGAAGATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCAT TGTGAATC CCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGGTGCGCATTCCTGGTGTGCTGCAGC GATTGGGAG TGACATACTTTGTGGTTGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATT GTGCCTCGG AGAGGAGCTGCCTTTCTCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCC TGGTGCTGG AAGGCCTGTGGCTGGGCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATC TTGGTCCTG GGGGCATTGGAGATTTTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCG ACCGCCTG CTGCTGGGAGACGATCACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAG GTGGCCTAT GACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAG GCAGGAAA AATACTATTGTATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTG TTGTATTCT TGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAA CAAAAATCT CTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCT GTACCCAGTT GTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATT CTGGTATA TGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCA GTCCCACA AGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCC TCTATAGAA AGAAGATTTTTTGGAAAATCTGAGTCGACACCCTGCCGCCTGGACTCCGCCTGTTAGAGC TCGCTGATC AGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTC CTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTG AGTAGGTGT CATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAAT AGCAGGC ATGCTGGGGAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC TCGCTCGC TCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAG TGAGCGAG CGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCCAGCTGCAT TAATGAAT CGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAC TGACTCGCT GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTT ATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAA AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC GACGCTCA AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGC TCCCTCGT GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG AAGCGTGGC GCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT GGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTC CAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTA TGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTT GGTATCTG CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA AACCACCG CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC AAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT TTGGTCATG AGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA ATCTAAAGT ATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA GCGATCTGT CTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG GGCTTACCA TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA GCAATAAA CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCA GTCTATTA ATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTG CCATTGCTA CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC GATCAAGGC GAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG TTGTCAGAA GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTG TCATGCCAT CCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA TGCGGCGA CCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTA AAAGTGCT CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATC CAGTTCGA TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTG GGTGAGCAA AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATAC TCATACT CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT ATTTGAATGT ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC GTCTAAGA AACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCT CGCGCGTTT CGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCT GTAAGCGG ATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCT GGCTTAA CTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCA CAGATGCG TAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTT AAATTTTTG TTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAA AGAATAGA CCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGG ACTCCAAC GTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAA TCAAGTTT TTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAG AGCTTGAC GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTA GGGCG CTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCG CTACAGGG CGCGTCGCGCCATTCGCCATTCAGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGC CTCTTCGC TATTACGCCAGCTGGCTGCA pTR-CMV-hHGSNAT (SEQ ID NO:3) GGGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACC AAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG AGAGGGA GTGGCCAACTCCATCACTAGGGGTTCCTAGATCTGAATTCGGTACCCGTTACATAACTTA CGGTAAATG GCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTC CCATAGTA ACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCAC TTGGCAGT ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC CGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTA TTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTT CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGAC TTTCCAAA ATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGT CTATATAA GCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACC TCCATAGA AGACACCGGGACCGATCCAGCCTCCGGACTCTAGAATGAGCGGGGCGGGCAGGGCGCTGG CCGCGCT GCTGCTGGCCGCGTCCGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCG CGATGCCC AGGCCGCGCCGCCACGAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGG CTTTGCT ACTCATCCATAATGAACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTG TTATCACTG CTTGTTTCAGGTTCTGGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGC AGCTGCCT CTGTCAGCACCCAGCACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAG TTTGTAGG TTGGAATACAGATTTGGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAAT GGAGTTAG TGAAATTGCCTGTGACCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAG CATTGCATT CCTTATTGGTCTTGCTGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGA TGACTTTAAC AATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTG GGATCTCCC AGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTG CCGCCCCG CCTCCGCAGCGTGGACACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTCAATTA TGGAGGAGG AAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTT CCCGTGGT TTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGT GTTCAAAATT CAGATTGCTGGGGAAGATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCAT TGTGAATC CCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGGTGCGCATTCCTGGTGTGCTGCAGC GATTGGGAG TGACATACTTTGTGGTTGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATT GTGCCTCGG AGAGGAGCTGCCTTTCTCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCC TGGTGCTGG AAGGCCTGTGGCTGGGCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATC TTGGTCCTG GGGGCATTGGAGATTTTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCG ACCGCCTG CTGCTGGGAGACGATCACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAG GTGGCCTAT GACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAG GCAGGAAA AATACTATTGTATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTG TTGTATTCT TGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAA CAAAAATCT CTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCT GTACCCAGTT GTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATT CTGGTATA TGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCA GTCCCACA AGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCC TCTATAGAA AGAAGATTTTTTGGAAAATCTGAGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCT TCTAGTTGC CAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC ACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGG GTGGGGTG GGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAGAGATCT AGGAAC CCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC GGGCAAAG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAG GGAGTGG CCAACCCCCCCCCCCCCCCCCCTGCAGCCCAGCTGCATTAATGAATCGGCCAACGCGCGG GGAGAGGC GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC GCAGGAA AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CGTTTTT CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG AAACCCG ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT CCGACCCTG CCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGC TCACGCTGT AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC GTTCAGCCC GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA TCGCCACTG GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTC TTGAAGT GGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGC CAGTTACC TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT TTTTTTGTT TGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCT ACGGGGTC TGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG GATCTTCA CCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAA CTTGGTCTG ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCAT CCATAGTTG CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG CTGCAATG ATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA AGGGCCG AGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGG AAGCTAGA GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTG GTGTCACGC TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA TCCCCCATG TTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC GCAGTGTTA TCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGC TTTTCTGTG ACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCT TGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA ACGTTCTT CGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTC GTGCACCC AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGG CAAAATGC CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCA ATATTATT GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA ATAAACAA ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATT ATCATGAC ATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGA CGGTGAAA ACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGA GCAGACAA GCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCA TCAGAGCA GATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAA ATACCGC ATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCA GCTCATTTTT TAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGG GTTGAGT GTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGG CGAAAAAC CGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTC GAGGTGCC GTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGC CGGCGAA CGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGT AGCGGT CACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCG CCATTCGC CATTCAGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCC AGCTGGCT GCA ptrsk-mCMV-hHGSNAT ZC (SEQ ID NO:4) CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT TCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATAC GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAA AGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGC ATCACAAA AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTT CCCCCTG GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT TTCTCCCTTC GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT TCGCTCCAA GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGA GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG CAGAGCG AGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGT ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG ATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGAT CTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGG ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAA TCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG GCACCTATC TCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT ACGATACGG GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCT CCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTG TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT CCGGTTCCC AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCG GTCCTCCG ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT AATTCTCTT ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC TGAGAATAG TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACAT AGCAGAAC TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGA GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCA CCAGCGTTT CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGA AATGTT GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA TGAGCGGATA CATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCAC CTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGA GGCCCTTT CGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG GTCACAGC TTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGG CGGGTGTC GGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGT GTGAAATA CCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGATTCCAACATCCAATAAATCAT ACAGGCA AGGCAAAGAATTAGCAAAATTAAGCAATAAAGCCTCAGAGCATAAAGCTAAATCGGTTGT ACCAAAA ACATTATGACCCTGTAATACTTTTGCGGGAGAAGCCTTTATTTCAACGCAAGGATAAAAA TTTTTAGAA CCCTCATATATTTTAAATGCAATGCCTGAGTAATGTGTAGGTAAAGATTCAAACGGGTGA GAAAGGCC GGAGACAGTCAAATCACCATCAATATGATATTCAACCGTTCTAGCTGATAAATTCATGCC GGAGAGGG TAGCTATTTTTGAGAGGTCTCTACAAAGGCTATCAGGTCATTGCCTGAGAGTCTGGAGCA AACAAGAG AATCGATGAACGGTAATCGTAAAACTAGCATGTCAATCATATGTACCCCGGTTGATAATC AGAAAAGC CCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAGCGTTAATATTTTGTTA AAATTCGC GTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCC TTATAAATC AAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATT AAAGAAC GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAA CCATCAC CCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGA GCCCCCGA TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAA GGAGCG GGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCG CTTAATGC GCCGCTACAGGGCGCGTACTATGGTTGCTTTGACGAGCACGTATAACGTGCTTTCCTCGT TAGAATCAG AGCGGGAGCTAAACAGGAGGCCGATTAAAGGGATTTTAGACAGGAACGGTACGCCAGAAT CCTGAGA AGTGTTTTTATAATCAGTGAGGCCACCGAGTAAAAGAGTCTGTCCATCACGCAAATTAAC CGTTGTCG CAATACTTCTTTGATTAGTAATAACATCACTTGCCTGAGTAGAAGAACTCAAACTATCGG CCTTGCTGG TAATATCCAGAACAATATTACCGCCAGCCATTGCAACGGAATCGCCATTCGCCATTCAGG CTGCGCAA CTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGCGCGCTCGCTC GCTCACTG AGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCG AGCGAGC GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGGAAGCTTTCGTTACATA ACTTACGG TAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGGACTCACGGGGATTTCCAAGTCT CCACCCCA TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA ACAACTCC GCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT CGTTTAGT GAACCGCTCGACCGGTCGCCATGAGCGGGGCGGGCAGGGCGCTGGCCGCGCTGCTGCTGG CCGCGTC CGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCGCGATGCCCAGGCCGC GCCGCCAC GAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGGCTTTGCTACTCATCC ATAATGA ACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTGTTATCACTGCTTGTT TCAGGTTCT GGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGCAGCTGCCTCTGTCAG CACCCAGC ACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAGTTTGTAGGTTGGAAT ACAGATTT GGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAATGGAGTTAGTGAAATT GCCTGTGA CCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAGCATTGCATTCCTTAT TGGTCTTGC TGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGATGACTTTAACAATTG GATTTCTAAA GCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGATCTCCCAGCAGG ACAGACCC TCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTGCCGCCCCGCCTCCG CAGCGTGGA CACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTCAATTATGGAGGAGGAAAATA TTGGTACTT CAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTTCCCGTGGTTTGTATT TATTATGGG ATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGTGTTCAAAATTCAGATT GCTGGGGAA GATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCATTGTGAATCCCAATTA TTGCCTTGG TCCATTGTCTTGGGACAAGGTGCGCATTCCTGGTGTGCTGCAGCGATTGGGAGTGACATA CTTTGTGGT TGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAG CTGCCTTTC TCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCT GTGGCTGG GCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCA TTGGAGATT TTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGG GAGACGAT CACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAGGTGGCCTATGACCCC GAGGGCATC CTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGGAAAAATACTA TTGTATTAC AAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTGTTGTATTCTTGGGCTC ATTTCTGTT GCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAACAAAAATCTCTGGTCC CTTTCGTAT GTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCTGTACCCAGTTGTGGAT GTGAAGGGG CTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATTCTGGTATATGTCGGC CACGAGGTG TTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCAGTCCCACAAGGAGCAC CTGACTCA GAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCCTCTATAGAAAGAAGAT TTTTTGGAA AATCTGACGTCGACACCCTGCCGCCTGGACTCCGCCTGTCATCAGATCTAAATAAAATAC GAAATG TTAAACCCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACG CCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCTG ptrsk-mCMV-hHGSNAT (SEQ ID NO:5) CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT TCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATAC GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAA AGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGC ATCACAAA AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTT CCCCCTG GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT TTCTCCCTTC GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT TCGCTCCAA GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGA GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG CAGAGCG AGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGT ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG ATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGAT CTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGG ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAA TCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG GCACCTATC TCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT ACGATACGG GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCT CCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTG TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT CCGGTTCCC AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCG GTCCTCCG ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT AATTCTCTT ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC TGAGAATAG TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACAT AGCAGAAC TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGA GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCA CCAGCGTTT CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGA AATGTT GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA TGAGCGGATA CATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCAC CTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGA GGCCCTTT CGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG GTCACAGC TTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGG CGGGTGTC GGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGT GTGAAATA CCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGATTCCAACATCCAATAAATCAT ACAGGCA AGGCAAAGAATTAGCAAAATTAAGCAATAAAGCCTCAGAGCATAAAGCTAAATCGGTTGT ACCAAAA ACATTATGACCCTGTAATACTTTTGCGGGAGAAGCCTTTATTTCAACGCAAGGATAAAAA TTTTTAGAA CCCTCATATATTTTAAATGCAATGCCTGAGTAATGTGTAGGTAAAGATTCAAACGGGTGA GAAAGGCC GGAGACAGTCAAATCACCATCAATATGATATTCAACCGTTCTAGCTGATAAATTCATGCC GGAGAGGG TAGCTATTTTTGAGAGGTCTCTACAAAGGCTATCAGGTCATTGCCTGAGAGTCTGGAGCA AACAAGAG AATCGATGAACGGTAATCGTAAAACTAGCATGTCAATCATATGTACCCCGGTTGATAATC AGAAAAGC CCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAGCGTTAATATTTTGTTA AAATTCGC GTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCC TTATAAATC AAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATT AAAGAAC GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAA CCATCAC CCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGA GCCCCCGA TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAA GGAGCG GGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCG CTTAATGC GCCGCTACAGGGCGCGTACTATGGTTGCTTTGACGAGCACGTATAACGTGCTTTCCTCGT TAGAATCAG AGCGGGAGCTAAACAGGAGGCCGATTAAAGGGATTTTAGACAGGAACGGTACGCCAGAAT CCTGAGA AGTGTTTTTATAATCAGTGAGGCCACCGAGTAAAAGAGTCTGTCCATCACGCAAATTAAC CGTTGTCG CAATACTTCTTTGATTAGTAATAACATCACTTGCCTGAGTAGAAGAACTCAAACTATCGG CCTTGCTGG TAATATCCAGAACAATATTACCGCCAGCCATTGCAACGGAATCGCCATTCGCCATTCAGG CTGCGCAA CTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGCGCGCTCGCTC GCTCACTG AGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCG AGCGAGC GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGGAAGCTTTCGTTACATA ACTTACGG TAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGGACTCACGGGGATTTCCAAGTCT CCACCCCA TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA ACAACTCC GCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT CGTTTAGT GAACCGCTCGACCGGTCGCCATGAGCGGGGCGGGCAGGGCGCTGGCCGCGCTGCTGCTGG CCGCGTC CGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCGCGATGCCCAGGCCGC GCCGCCAC GAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGGCTTTGCTACTCATCC ATAATGA ACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTGTTATCACTGCTTGTT TCAGGTTCT GGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGCAGCTGCCTCTGTCAG CACCCAGC ACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAGTTTGTAGGTTGGAAT ACAGATTT GGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAATGGAGTTAGTGAAATT GCCTGTGA CCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAGCATTGCATTCCTTAT TGGTCTTGC TGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGATGACTTTAACAATTG GATTTCTAAA GCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGATCTCCCAGCAGG ACAGACCC TCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTGCCGCCCCGCCTCCG CAGCGTGGA CACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTCAATTATGGAGGAGGAAAATA TTGGTACTT CAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTTCCCGTGGTTTGTATT TATTATGGG ATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGTGTTCAAAATTCAGATT GCTGGGGAA GATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCATTGTGAATCCCAATTA TTGCCTTGG TCCATTGTCTTGGGACAAGGTGCGCATTCCTGGTGTGCTGCAGCGATTGGGAGTGACATA CTTTGTGGT TGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAG CTGCCTTTC TCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCT GTGGCTGG GCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCA TTGGAGATT TTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGG GAGACGAT CACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAGGTGGCCTATGACCCC GAGGGCATC CTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGGAAAAATACTA TTGTATTAC AAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTGTTGTATTCTTGGGCTC ATTTCTGTT GCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAACAAAAATCTCTGGTCC CTTTCGTAT GTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCTGTACCCAGTTGTGGAT GTGAAGGGG CTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATTCTGGTATATGTCGGC CACGAGGTG TTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCAGTCCCACAAGGAGCAC CTGACTCA GAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCCTCTATAGAAAGAAGAT TTTTTGGAA AATCTGACCATCAGATCTAAATAAAATACGAAATGTTAAACCCCACTCCCTCTCTGCGCG CTCGCTCGC TCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG TGAGCGA GCGAGCGCGCAGCTGCTG ptrsk-CMV-hSMN1 ZC (SEQ ID NO:6) CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCG ACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGGTTTAAACCTAAAAA ACCTCCC ACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTAT TGCAGCTTA TAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACT GCATTCTAG TTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGAGATCTACAGGCGGAGTC CAGGCGGCA GGGTTTAATTTAAGGAATGTGAGCACCTTCCTTCTTTTTGATTTTGTCTGAAACCCATAT AATAGCCAG TATGATAGCCACTCATGTACCATGAAATTAACATACTTCCCAAAGCATCAGCATCATCAA GAGAATCT GGACATATGGGAGGTGGTGGGGGAATTATTGGTGGTCCAGAAGGAAATGGAGGCAGCCAG CATGATA GTAAGTGGGGTGGTGGTGGTGGCGGTGGCGGTGGTGGGCCATTGAATTTTAGACCTGGCT TTCCTGGT CCCAGTCTTGGCCCTGGCATGGGGGGTGGTGGAGGGAGAAAAGAGTTCCATGGAGCAGAT TTGGGCTT GATGTTATCTGATTTATTTCCAGGAGACCTGGAGTTCTCACTTTCATCTGTTGAAACTTG GCTTTCATTT TCATTCTCTTGAGCATTTTGTTCTATATTATTAGCTACTTCACAGATTGGGGAAAGTAGA TCGGACAGA TTTTGCTCCTCTCTATTTCCATATCCAGTGTAAACCACAACACAGGTTTCTCTCTTAAAA TCAATTGAAG CAATGGTAGCTGGGTAAATGCAACCGTCTTCTGACCAAATGGCAGAACATTTGTCCCCAA CTTTCCACT GTTGTAAGGAAGCTGCAGTATTCTTCTTTTGGCTTTTATTCTTCTTAGCAGGTTTTCTTT TAGGTGTGGT TTTTGGTTTACCCGAAGTTTCACAAATGTCACCATTCTTTAGAGCATGCTTAAATGAAGC CACAGCTTT ATCATATGCTTTTATCAGTGCTGTATCATCCCAAATGTCAGAATCATCGCTCTGGCCTGT GCCGCGCCG GAACAGCACGGAATCCTCCTGCTCCGGGACGCCGCCACCACTGCCGCCGCTGCTCATCGC CATAccggT CCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCT CCAGGCGA TCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGC CCATTTGCG TCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACA AACTCCCA TTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCA TTGATGTA CTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGT AGGAAAGT CCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAA TAGGGGGC GTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCC ACCCATTGA CGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAAT GGGCGGGG GTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGAagcttCCTAG GAACCCCTAG TGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAA AGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGGCGTAAT AGCGAAG AGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGcgattCC GTTGCAATGG CTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTC AGGCAAGTG ATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTC TTTTACTCG GTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAA TCCCTTTAA TCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCG TCAAAGCAA CCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC GTGACCGC TACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCAC GTTCGCCGGC TTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGG CACCTCGAC CCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT TTTCGCCCT TTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATC TCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT GAGCTGATTT AACAAAAATTTAACGCGAATTTTAACAAAATATTAACGcTTACAATTTAAATATTTGCTT ATACAATCT TCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTT TACGATTACC GTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGA GACCTCTCA AAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGAT GGTGATTTG ACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCA TTTAAAATAT ATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTAT TACAGGGTC ATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTG CTAATTCTTT GCCTTGCCTGTATGATTTATTGGATGTTGGAATcgCCTGATGCGGTATTTTCTCCTTACG CATCTGTGCG GTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGCCCC GACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT ACAGACAA GCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGC GCGAGACG AAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTA GACGTCAGG TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTC AAATATGTA TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT GAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC ACCCAGAAAC GCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT GGATCTCA ACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTC TGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCA TACACTAT TCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATG ACAGTAAG AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC AACGATCG GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTG ATCGTTGG GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCA ATGGCAA CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA TAGACTGG ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT ATTGCTGAT AAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGT AAGCCCTC CCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA GATCGCTG AGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATAC TTTAGATTG ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA TGACCAAAA TCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAG ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG TGGTTTGTT TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGA TACCAAAT ACTGTtCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT ACATACCTC GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG TTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG CCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA CGCTTCC CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCAC GAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT TGAGCGTCG ATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTT TTTACGGT TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG TGGATAACCG TATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGA GTCAGTGA GCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTC ATTAATG ptrsk-CMV-hSMN1 (SEQ ID NO:7) CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCG ACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGGTTTAAACCTAAAAA ACCTCCC ACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTAT TGCAGCTTA TAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACT GCATTCTAG TTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGAGATCTTTAATTTAAGGA ATGTGAGCA CCTTCCTTCTTTTTGATTTTGTCTGAAACCCATATAATAGCCAGTATGATAGCCACTCAT GTACCATGA AATTAACATACTTCCCAAAGCATCAGCATCATCAAGAGAATCTGGACATATGGGAGGTGG TGGGGGA ATTATTGGTGGTCCAGAAGGAAATGGAGGCAGCCAGCATGATAGTAAGTGGGGTGGTGGT GGTGGCG GTGGCGGTGGTGGGCCATTGAATTTTAGACCTGGCTTTCCTGGTCCCAGTCTTGGCCCTG GCATGGGGG GTGGTGGAGGGAGAAAAGAGTTCCATGGAGCAGATTTGGGCTTGATGTTATCTGATTTAT TTCCAGGA GACCTGGAGTTCTCACTTTCATCTGTTGAAACTTGGCTTTCATTTTCATTCTCTTGAGCA TTTTGTTCTAT ATTATTAGCTACTTCACAGATTGGGGAAAGTAGATCGGACAGATTTTGCTCCTCTCTATT TCCATATCC AGTGTAAACCACAACACAGGTTTCTCTCTTAAAATCAATTGAAGCAATGGTAGCTGGGTA AATGCAAC CGTCTTCTGACCAAATGGCAGAACATTTGTCCCCAACTTTCCACTGTTGTAAGGAAGCTG CAGTATTCT TCTTTTGGCTTTTATTCTTCTTAGCAGGTTTTCTTTTAGGTGTGGTTTTTGGTTTACCCG AAGTTTCACAA ATGTCACCATTCTTTAGAGCATGCTTAAATGAAGCCACAGCTTTATCATATGCTTTTATC AGTGCTGTA TCATCCCAAATGTCAGAATCATCGCTCTGGCCTGTGCCGCGCCGGAACAGCACGGAATCC TCCTGCTC CGGGACGCCGCCACCACTGCCGCCGCTGCTCATCGCCATACCGGTCCGGAGGCTGGATCG GTCCCGGT GTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTA AACGAGCT CTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAG TTGTTACGA CATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGG TGGAGACT TGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCAT CACCATGG TAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGT ACTGGGCA TAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGA TACACTTG ATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGT CCCTATTG GCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTC AGCCAGGC GGGCCATTTACCGTAAGTTATGTAACGAAGCTTCCTAGGAACCCCTAGTGATGGAGTTGG CCACTCCC TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGC TTTGCCCG GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA TCGCCCT TCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGATTCCGTTGCAATGGCTGGCGGTAA TATTGTTC TGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTA CTAATCAAA GAAGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCA CTGATTATA AAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCC TGTTTAGCT CCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTAC GCGCCCTG TAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC CAGCGCCC TAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCT AAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA ACTTGATTA GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTT GGAGTCCAC GTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTA TTCTTTTGAT TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAA TTTAACGCG AATTTTAACAAAATATTAACGCTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTT TTGGGGCTTT TCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCG ATTCTCTTGT TTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCT ACCCTCTCC GGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCC GGCCTTTCT CACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGT TCTAAAAAT TTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTT TTTGGTACA ACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGC CTGTATGATTT ATTGGATGTTGGAATCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCA CACCGCATA TGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCG CCAACACC CGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGAC CGTCTCCG GGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCC TCGTGATA CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT TTTCGGGGA AATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC ATGAGACAA TAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTC CGTGTCGC CCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT GAAAGTAAAA GATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGT AAGATCCT TGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATG TGGCGCGGT ATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAA TGACTTGG TTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTAT GCAGTGCT GCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCG AAGGAGCT AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGA GCTGAATG AAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGC GCAAACT ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGC GGATAAAG TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTG GAGCCGGTG AGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCG TAGTTATC TACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGT GCCTCAC TGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAA AACTTCATT TTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGT TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT TTTTTCTGC GCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG ATCAAGAG CTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTT CTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTG CTAATCCTG TTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGA TAGTTACC GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG AACGACC TACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGG AGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG GGGGAA ACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT TGTGATGCTC GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGC CTTTTGCT GGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGA GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGA AGCGGAA GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG