LINDSAY MARK EVAN (US)
KLEINSTIVER BENJAMIN P (US)
MAGUIRE CASEY A (US)
CHUNG DAVID YOUNG (US)
CARDENAS CHRISTIAN LACKS LINO (US)
MALHOTRA RAJEEV (US)
WHAT IS CLAIMED IS: 1. An isolated nucleic acid comprising a promoter sequence that has at least 90% identity to SEQ ID NO: 4 and optionally contains at least one substitution modification relative to SEQ ID NO: 4, or a promoter sequence that has at least 90% identity to SEQ ID NO: 5 and optionally contains at least one substitution modification relative to SEQ ID NO: 5, or a promoter sequence that has at least 90% identity to SEQ ID NO: 6 and optionally contains at least one substitution modification relative to SEQ ID NO: 6, or a promoter sequence that has at least 90% identity to SEQ ID NO: 85 and optionally contains at least one substitution modification relative to SEQ ID NO: 85, or a promoter sequence that has at least 90% identity to SEQ ID NO: 86 and optionally contains at least one substitution modification relative to SEQ ID NO: 86, or a promoter sequence that has at least 90% identity to SEQ ID NO: 87 and optionally contains at least one substitution modification relative to SEQ ID NO: 87. 2. An isolated nucleic acid comprising a heterologous transgene and a promoter sequence that has at least 90% identity to SEQ ID NO: 4, 5, 6, 85, 86, or 87, preferably wherein the promoter sequence is operably linked to the heterologous coding sequence. 3. The isolated nucleic acid of claim 1 or 2, wherein the promoter drives transcription of the heterologous coding sequence in vascular smooth muscle cells. 4. A vector comprising the nucleic acid of claim 1, and a heterologous nucleic acid sequence, optionally a transgene operably linked to the promoter sequence, optionally wherein the vector is encapsulated by an adeno-associated virus (AAV) capsid. 5. An adeno-associated virus (AAV) vector comprising (i) one or more of peptides selected from the group consisting of PRPPSTH (SEQ ID NO:1), MAEPGAR (SEQ ID NO:25), MLYADNT (SEQ ID NO: 26), and SQDPSTL (SEQ ID NO: 27), in its capsid, and (ii) a nucleic acid sequence comprising a smooth muscle cell-specific promoter or active portion thereof encapsulated by the capsid. 6. The AAV vector of claim 5, wherein the smooth muscle cell-specific promoter is an HDAC9-derived promoter. 7. The AAV vector of claim 6, wherein the HDAC9-derived promoter comprises a sequence that has at least 90% identity to SEQ ID NO: 4, 5, 6, 85, 86, or 87. 8. The AAV vector of any of claims 5-7, wherein the smooth muscle promoter is operably linked to a heterologous transgene. 9. The AAV vector of claim 8, wherein the transgene encodes a genome editor or an intein- split construct thereof, optionally selected from CRISPR nucleases, cytosine base editors (CBEs), adenine base editors (ABEs), and CRISPR prime editors (PEs). 10. The AAV vector of claim 9, wherein the transgene encodes: (a) a CRISPR/Cas base editor or an intein-split construct thereof, and/or an Arg179His mutant-allele specific guide RNA directing a base editor to the mutation, and/or an Arg179Cys mutant-allele specific guide RNA directing a base editor to the mutation, or (b) a CRISPR/Cas genome editor or an intein-split construct thereof, and/or an Arg179His mutant-allele specific guide RNA directing a genome editor to the mutation, and/or an Arg179Cys mutant-allele specific guide RNA directing a base editor to the mutation. 11. The AAV vector of claim 5, wherein the sequence PRPPSTH (SEQ ID NO: 1) is inserted into VP1 protein of the AAV vector. 12. The AAV vector of claim 11, wherein the sequence PRPPSTH (SEQ ID NO: 1) is inserted into the VP1 protein in a position corresponding to amino acids 588 and 589 of SEQ ID NO: 2. 13. The AAV vector of claims 5-12, wherein the AAV serotype is AAV-9 or AAV-2. 14. A composition comprising the vector of claim 4 or the AAV vectors of claims 5-13. 15. A recombinant episome comprising a nucleic acid sequence that encodes an adeno- associated virus (AAV) capsid having one or more of peptides selected from the group consisting of PRPPSTH (SEQ ID NO:1), MAEPGAR (SEQ ID NO:25), MLYADNT (SEQ ID NO: 26), and SQDPSTL (SEQ ID NO: 27) in its capsid, and a nucleic acid sequence comprising a smooth muscle specific promoter or active portion thereof encapsulated by the capsid. 16. The recombinant episome of claim 15, wherein at least two copies of the one or more of peptides selected from the group consisting of PRPPSTH (SEQ ID NO:1), MAEPGAR (SEQ ID NO:25), MLYADNT (SEQ ID NO:26), and SQDPSTL (SEQ ID NO:27) are inserted into the capsid of the AAV vector. 17. The recombinant episome of claim 16, wherein the at least two copies are of a same peptide. 18. The recombinant episome of claim 16, wherein the at least two copies are of a different peptide. 19. The recombinant episome of claim 15, wherein the one or more of peptides selected from the group consisting of PRPPSTH (SEQ ID NO:1), MAEPGAR (SEQ ID NO:25), MLYADNT (SEQ ID NO:26), and SQDPSTL (SEQ ID NO:27) are inserted into the VP1 protein of the AAV vector. 20. The recombinant episome of claim 19, wherein the one or more of peptides selected from the group consisting of PRPPSTH (SEQ ID NO:1), MAEPGAR (SEQ ID NO:25), MLYADNT (SEQ ID NO: 26), and SQDPSTL (SEQ ID NO: 27) are inserted into the VP1 protein of the AAV vector in a position corresponding to amino acids 588 and 589 of SEQ ID NO: 2. 21. The recombinant episome of claims 15 to 19, wherein the AAV serotype is AAV-9 or AAV-2. 22. A composition comprising the AAV vector encoded by the recombinant episome of claims 15 to 21. 23. A method of correcting an Arg179His mutation or an Arg179Cys mutation in the genome of a cell, the method comprising delivering to the cell: (i) a CRISPR/Cas base editor or an intein-split construct thereof, and an Arg179His or an Arg179Cys mutant-allele specific guide RNA directing the base editor to the mutation, or (ii) a CRISPR/Cas genome editor or an intein-split construct thereof, and an Arg179His or an Arg179Cys mutant-allele specific guide RNA directing the genome editor to the mutation. 24. The method of claim 23, wherein the base editor and/or guide RNA are listed in Table 1 and/or Table 2 (comprising SEQ ID NOs:55-79), optionally comprising SEQ ID NO:60. 25. The method of claim 23, wherein the Arg179His or the Arg179Cys mutant-allele specific CRISPR/Cas base editor is an adenine base editor or an intein-split construct thereof comprising the wild-type SpCas9, A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R1333P/R1335 Q/T1337R (SpRY), D10T(optional)/I322V/S409I/E427G/R654L/R753G/R1114G/D1135N/V1139A/D1180G/ E1219V/Q1221H/A1320V/R1333K (SpCas9-NRRH), D10T(optional)/I322V/S409I/E427G/R654L/R753G/R1114G/D1135N/E1219V/D1332N/ R1335Q/T1337N/S1338T/H1349R (SpCas9-NRCH), D1135M/S1136Q/G1218K/E1219S/R1335E/T1337R (MQSKER), D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R (SpG) or D1135V/G1218R/R1335Q/T1337R (VRQR) or S55R/D1135V/G1218R/R1335Q/T1337R (VRQR(S55R)) variants of Streptococcus pyogenes Cas9 protein (SpCas9). 26. The method of any of claims 23 to 25, wherein the spacer sequence of the guide RNA targets the sequence TGCATCTGGATCTGGCTGGC (SEQ ID NO:28; HES1208-A4 gRNA), optionally in combination with ABE8e-SpCas9-VRQR, ABE8e-SpCas9- VRQR(S55R), ABE8e-SpG, ABE8e-SpRY, or ABE8e-SpCas9-NRRH, or an intein-split construct thereof; the spacer sequence of the guide RNA targets the sequence TCATGCATCTGGATCTGGCT (SEQ ID NO: 24, HES1210-A7 gRNA), optionally in combination with ABE8e-SpG, ABE8e-SpRY, ABE8e-SpCas9-NRCH, ABE8e-MQSKER or an intein-split construct thereof; or the spacer sequence of the guide RNA targets the sequence ATCATGCATCTGGATCTGGC (SEQ ID NO:23, HES1212-A8), optionally in combination with ABE8e-SpCas9, ABE8e-SpG, ABE8e-SpRY, or ABE8e-SpCas9-NRRH or an intein-split construct thereof. 27. The method of claim 23 or 24, wherein the genome editor is a wild-type SpCas9 or an intein-split construct thereof. 28. The method of claim 27, wherein the spacer sequence of the guide RNA comprises the sequence TGCCATCATGCATCTGGATC (HES1235, SEQ ID NO:83) or AGCCAGATCCAGATGCATGA (HES1236, SEQ ID NO:84). 29. The method of any of claims 23 to 28, wherein the base editor or intein-split construct thereof and guide RNA, or the genome editor or intein-split construct thereof and guide RNA, are delivered to the cell using one or more adeno-associated virus (AAV) vectors, preferably AAV comprising the sequence PRPPSTH (SEQ ID NO:1) inserted into the VP1 protein, in a position corresponding to between amino acids 588 and 589 of SEQ ID NO:2, and preferably comprising an HDAC9 promoter or active portion thereof. 30. The method of claim 29, wherein the HDAC9 promoter comprises a sequence that has at least 90% identity to SEQ ID NO: 4, 5, 6, 85, 86, or 87. 31. The method of any of claims 23 to 30, wherein the cell is in a living subject. 32. The method of claim 31, wherein the living subject is a human with: (i) an Arg179His mutation in an allele of ACTA2, and the method comprises delivering a base editor or intein-split construct thereof and/or guide RNA, or (ii) an Arg179Cys mutation in an allele of ACTA2, and the method comprises delivering a genome editor or intein-split construct thereof and/or guide RNA. 33. A method of treating a vasculopathy, optionally multisystem smooth muscle dysfunction syndrome (MSMDS), in a subject who has an ACTA2 R179H mutation, the method comprising delivering to the vasculature of the subject a therapeutically effective amount of a gene therapy agent comprising: (a) a CRISPR/Cas base editor or an intein-split construct thereof, and a guide RNA directing the base editor to the Arg179His mutation, or (b) a CRISPR/Cas genome editor or an intein-split construct thereof, and a guide RNA directing the genome editor to the Arg179His mutation. 34. The method of claim 33, wherein the base editor or intein-split construct thereof, genome editor or intein-split construct thereof, and/or guide RNA are listed in Table 1 and/or Table 2(comprising SEQ ID NOs:55-79), optionally comprising SEQ ID NO:60. 35. The method of claim 33 or 34, wherein the Arg179His mutant-allele specific CRISPR/Cas base editor is an adenine base editor or an intein-split construct thereof comprising the wild type Cas9, A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R1333P/R1335 Q/T1337R (SpRY), D10T(optional)/I322V/S409I/E427G/R654L/R753G/R1114G/D1135N/V1139A/D1180G/ E1219V/Q1221H/A1320V/R1333K (SpCas9-NRRH), D10T(optional)/I322V/S409I/E427G/R654L/R753G/R1114G/D1135N/E1219V/D1332N/ R1335Q/T1337N/S1338T/H1349R (SpCas9-NRCH), D1135M/S1136Q/G1218K/E1219S/R1335E/T1337R (MQSKER), D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R (SpG) or D1135V/G1218R/R1335Q/T1337R (VRQR) or S55R/D1135V/G1218R/R1335Q/T1337R (VRQR(S55R)) variants of Streptococcus pyogenes Cas9 protein (SpCas9). 36. The method of any of claims 33 to 35, wherein the spacer sequence of the guide RNA directing the base editor to the mutation comprises the sequence TGCATCTGGATCTGGCTGGC (SEQ ID NO:28, HES1208-A4 gRNA), optionally in combination with ABE8e-SpCas9-VRQR, ABE8e-SpCas9-VRQR(S55R), ABE8e-SpG, ABE8e-SpRY, or ABE8e-SpCas9-NRRH or an intein-split construct thereof; the spacer sequence of the guide RNA targets the sequence TCATGCATCTGGATCTGGCT (SEQ ID NO: 24, HES1210-A7 gRNA), optionally in combination with ABE8e-SpG, ABE8e- SpRY, ABE8e-SpCas9-NRCH, ABE8e-MQSKER or an intein-split construct thereof; or the spacer sequence of the guide RNA targets the sequence ATCATGCATCTGGATCTGGC (SEQ ID NO:23, HES1212-A8), optionally in combination with ABE8e-SpCas9, ABE8e-SpG, ABE8e-SpRY, or ABE8e-SpCas9-NRRH or an intein-split construct thereof. 37. The method of claim 33, wherein the genome editor and/or guide RNA are listed in Table 1 and/or Table 2 (comprising SEQ ID NOs:55-79), optionally comprising SEQ ID NO:60. 38. The method of claim 37, wherein the genome editor is a wild-type SpCas9 or an intein- split construct thereof. 39. The method of any of claims 33, 37, or 38, wherein the spacer sequence of the guide RNA directing the genome editor to the mutation comprises the sequence TGCCATCATGCATCTGGATC (HES1235, SEQ ID NO:83) or AGCCAGATCCAGATGCATGA (HES1236, SEQ ID NO:84). 40. The method of any of claims 33 to 39, wherein the base editor or intein-split construct thereof and guide RNA are delivered to the cell using adeno-associated virus (AAV) vectors, preferably AAV comprising the sequence PRPPSTH (SEQ ID NO:1) inserted into the VP1 protein, in a position corresponding to between amino acids 588 and 589 of SEQ ID NO:2, and preferably comprising an HDAC9-derived promoter. 41. The method of claim 40, wherein the HDAC9-derived promoter comprises a sequence that has at least 90% identity to SEQ ID NO: 4, 5, 6, 85, 86, or 87. 42. The method of any of claims 33 to 41, wherein the gene therapy agent is administered to the subject by local or systemic delivery. 43. The method of claim 42, wherein the gene therapy agent is administered systemically by intravenous or intra-arterial delivery. |
V. Methods of Treatment [00143] Provided herein are methods of treating vasculopathies, including subjects who have genetic or acquired vasculopathies, by delivering a therapeutically effective amount of a gene therapy agent. The disclosure demonstrates effective delivery of a therapy comprising a base editor for correcting a mutation known to drive an exemplary genetic vasculopathy, and of a nuclease to delete mutant alleles. The gene therapy agent (e.g., a CRISPR Cas nuclease specifically targeting an R179H mutant allele without or with a repair template encoding a replacement nucleic acid, for treating vasculopathies in subjects who have an ACTA2 R179H mutation) can be delivered in a viral vector, e.g., an AAV as described herein, preferably AAV-PR, preferably wherein expression is driven by an HDAC9- derived promoter as described herein. The vector can be delivered by any suitable route, e.g., locally (e.g., by intraocular, intravesical, or intrathecal delivery) or systemically (e.g., by systemic intravenous delivery). [00144] The present methods can be used to prevent (i.e., reduce the risk of) or delay progression of cerebral vasculopathy, white matter injury and strokes, e.g., in children without critical stenosis of the internal carotid artery (>70% narrowing between the petrous and clinoid ICA diameter). The present methods can also be used to prevent or delay dissection/aortic replacement surgery, e.g., in older children and young adults with high level of neurological function developing rapid progression of aortic dilatation. The present methods can also be used to treat multisystemic smooth muscle dysfunction syndrome (eye, lungs, bladder, gut) which can lead to life-threatening complications in this disease. In some embodiments, a treatment as described herein will lead to normalization of systemic blood pressure and positional orthostatic tachycardia. [00145] In some embodiments, on the cellular level, the present methods lead to normalization of F/G actin ratio (e.g., detectable by western blot and cell immunofluorescence); of gene expression profile (e.g., detectable by mRNA sequencing or PCR); or of Migration, Contractility and Proliferation (e.g., detectable by assays described herein or known in the art). VI. Mouse and Cellular Models of ACTA2 R179H MSMDS [00146] Also described herein are mouse models and several cellular models of MSMDS, including a conditional knock-in Acta2R179H mouse that when crossed with a Wnt1-Cre and Myh11-Cre mouse develops the characteristic SMC systemic dysregulation and early onset of aortic dilatation, cerebral steno-occlusive vasculopathy and BBB dysfunction. Also described is an ACTA2 R179H HEK 293T cell-line that permits evaluation of corrective genome editing strategies. These cellular models were used to demonstrate effective correction of the ACTA2179 codon from the disease causing ‘CAT’ codon to the wild type ‘CGT,’ allele-specific knockout of the R179H allele using CRISPR-Cas nucleases. This strategy was optimize for effective viral-mediated neurovascular delivery of nucleic acids and selectively editing the CAT to CGT codon, mimicking the needed pharmacologic delivery strategy in patients. [00147] Provided herein are conditional knock in ACTA2 Arg179His mouse models, the somatic and germ line cells of which include a mutant ACTA2 exon 6. The wild type allele is with a poly A terminator is surrounded by loxP sites. When the mice are crossed with a mouse expressing a Cre recombinase the wild type exon is removed and the mutant exon is spliced into the ACTA2 mRNA allele, resulting in expression of a mutant alpha smooth muscle actin within cell types expressing a Cre recombinase. For example, to activate the R179H allele in smooth muscle containing organs a cre recombinase driven by a smooth muscle cell myosin heavy chain promoter (Myh11-cre) or an HDAC9 promoter as described herein can be used. To express the mutant allele within the anterior cerebral circulation without other systemic effects, a neural crest specific cre allele (Wnt1-cre) 20 can be used. [00148] Also provided herein are mammalian, e.g., human, cell lines that include an ACTA2 Arg179His or ACTA2 Arg179Cys mutant allele in their genomic DNA. Exemplary cell lines include HEK293, HekT293, CHO. EXAMPLES [00149] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [00150] Materials and Methods [00151] The following materials and methods were used in examples described herein. [00152] Generation of the knock-in Acta2 R179H mouse model: All mice were cared for under strict compliance with the Partners Institutional Animal Care and Use Committee (IACUC), regulated by the United States Department of Agriculture and the United States Public Health Service and Institutional Animal Care and Use Committee of Massachusetts General Hospital, MA, USA. The Acta2 R179Hfl/+ mice were generated by subcontracting Cyagen company services (cyagen.com). Homozygous Acta2 R179Hfl/ R179Hfl mice were crossed with either Myh11-cre (all smooth muscle) or Wnt1-cre (neural crest smooth muscle) for activation of the mutation. The B6.Cg-Tg(Myh11-cre,-EGFP)2Mik/J (cat# 007742) and 129S4.Cg-E2f1Tg(Wnt1-cre)2Sor/J mice were purchased from Jackson laboratories. Both mouse lines were crossed to induce the expression of the Acta2 H179 mutant protein. For whole body histology mouse carcasses were imbedded in Buins fixative solution for >48hrs and brain and organs dissected, paraphing mounted and processed for conventional staining techniques including Hematoxylin Eosin (H&E), Luxol Fast (modified Kluver’s myelin sheath stanning), Trichrome and Cersyl Violet. In addition, for immunofluorescence analysis, fixed aortas and brains were cryopreserved with sucrose and embed in OCT or fixed with formalin 4%. Both male and female mice were included in all analysis (ratio 1:1). [00153] Gross Organ images: For aortas and neurovascular circulation, latex was injected into the left ventricular apex under low pressure until it was visible in the femoral artery. Animals were then fixed in Formalin (10%) for 24 h before transfer to 70% ethanol for dissection, photography, and storage. Other organs were then resected. [00154] Mouse aortic SMCs: SMCs were isolated by standard explant of the ascending section of the aortas from Acta2 R179Hfl/+ or Myh11-Cre: Acta2 R179Hfl/+ mice. In order to preserve cell identity, all experiments were carried out at passages 1–5. Human and murine SMCs were grown with SMC growth medium from Cell Applications Inc. (catalog 311-500). [00155] Histology: Organs were then removed from the animals or dissected in situ for photography prior to paraffinization and sectioning (7 ^M). Slides were produced for tissue staining or stained with standard stains including Elastin (Verhoeff-Van Gieson, Thermo Scientific, MI, USA) or F-actin (ActinGreen™ 488 ReadyProbes, ThermoFisher Scientific, USA) for quantitative analysis. For sequential fluorescence in situ hybridization and immunofluorescence microscopy aortas from human and mice were cryosectioned using OCT standard protocol. [00156] Smooth muscle cell Immunofluorescence: Smooth muscle cell identity was assessed by immunofluorescence staining of contractile markers including Myh11 (Abcam, ab53219), a-SMA (Abcam, ab5694), Sm22Į (Abcam, ab14106), Calponin1 (Abcam, ab46794), Smoothelin1 (Santa Cruz, sc-73042), and Vinculin1 (Abcam, ab18058). Cytoplasmic protein lysates were prepared using NE-PER Kit (Pierce, Rockford, IL, USA) and supplemented with 1ul~ of protease inhibitor cocktail (Roche) according to the manufacturer’s instruction. Mice were euthanized and perfused with 1X PBS. Brains were dissected and snap frozen in liquid nitrogen. Brains were kept at -80°C until ready to use. Coronal and sagittal brain sections (8 ^m, 20 ^m and 50 ^m) starting at the olfactory bulb were prepared sequentially at using a Leica cryostat. Sections were left to dry for 15 minutes minimum before storage at -80°C until needed for staining. [00157] Cresyl Violet Staining: Brain sections were stained in 0.1% cresyl violet solution for 10 minutes. Following thus sections were rinsed quickly in distilled water. Differentiate in 95% ethyl alcohol for 2 minutes followed by dehydration in 100% alcohol twice for 5 mins each. Mount with permanent mounting medium and image. [00158] Thioflavin Staining: Brain coronal sections were incubated in fresh filtered 1% Thioflavin-S solution for 8 minutes, at room temperature. Sections were washed twice for 3 mins each in 80% ethanol followed by a single wash in 95% ethanol for 3 min. Sections were washed with 3 exchanges of distilled water. Cover slipping was performed with mounting media and allowed to dry overnight. Following day the slides with coverslip was sealed with clear nail polish and imaged within a week. [00159] Ultrasounds: Nair hair removal cream was used on all mice the day prior to ultrasounds. All ultrasounds were performed on awake, unsedated mice using the Visualsonics Vevo660 imaging system and a 30 MHz transducer. The aorta was imaged using a standard parasternal long axis view. Dimensions from each animal represent averages of measurements made on still frames in systole of the maximal internal diameter of the aortic valve annulus, aortic sinuses, sinotubular junction, or ascending aorta by a cardiologist blinded to genotype. [00160] Neuroimaging Window Placement: A chronic glass coverslip was placed as previously described. 42 In brief, the surface of the skull was cleared of any residual blood products or debris. A glass coverslip (12ௗmm diameter, Electron Microscopy Sciences, emsdiasum.com, Cat #72196-12) was cut with a diamond pen to approximate the shape of the dorsal skull surface. C&B Metabond cement was mixed using one scoop of Clear L-Powder, six drops of Quick Base, and oneௗdrop of catalyst (Product numbers S399, S398, and S371, Parkell, Edgewood, NY, USA) in a ceramic mixing dish pre-chilled on ice. The cement was used to adhere the glass coverslip to the exposed skull. Additional cement was applied to fill any remaining gaps between the skull and coverslip. The cement was cured for an additional 15ௗmin and the animal allowed to recover. [00161] Resting state functional connectivity (RSFC): RSFC was performed at indicated time points. For each imaging session, mice were anesthetized with 2,2,2- tribromoethanol (TBE), also known as Avertin, which provides excellent RSFC signals. 43 Injectable TBE was prepared by mixing 0.5ௗmL stock solution (10ௗg 2,2,2-tribromoethanol in 6.25ௗmL tert-amyl alcohol; Sigma-Aldrich, T48402-25G, and Fisher Scientific, A730-1, respectively) with 39.5ௗmL 0.9% normal saline, stored at 4°C for no longer than one month, protecting from light at all stages. To ensure a smooth induction, TBE was injected in two to three divided doses intraperitoneally (0.3–0.4ௗmL initial dose, 0.1ௗmL at approximately 8ௗmin, and 0.1ௗmL at approximately 12ௗmin). More TBE was injected if the mouse did not achieve an adequate level of anesthesia to suppress withdrawal to toe pinch. The animal was placed on a homeothermic heating pad (37.0ௗ±ௗ0.1°C) and head-fixed in a stereotaxic frame. The glass coverslip was cleaned with cotton-tipped applicators and diluted ethanol solution. Image acquisition typically began at 18–20ௗmin after the initial dose of TBE. [00162] The acquisition and processing of single wavelength functional optical intrinsic signal imaging have been described previously. 44,45 The imaging surface was illuminated with a quartz tungsten halogen lamp (Techniquip R150, Capra Optical, Natick, MA) filtered at 570ௗ±ௗ10ௗnm and directed with a fiber optic cable. ^Manager software was used for image acquisition. 46 Images were acquired with a Cascade 512ௗF camera (Photometrics) at 512ௗ×ௗ512 pixel resolution, 11.1 FPS, and an exposure time of 50 ms for 12ௗmin. A custom script was written in MATLAB (Math Works, Natick, MA, USA) for image processing. The image was down-sampled to 256ௗ×ௗ256 pixels. Based on measurements from the Paxinos and Franklin mouse atlas, the final scale is 47 ^m/pixel. The optical density of each pixel over time was calculated and the density maps filtered between 0.008ௗHz and 0.09ௗHz. The signal was downsampled to 1 FPS. A brain mask was selected. The global signal was determined by taking the mean optical density for each frame. This global signal was then regressed from the optical density maps frame-by-frame over time. Imaging data were analyzed in a blinded manner. Seed-based connectivity maps were created by mapping the correlation coefficients between seeds placed in motor, somatosensory, retrosplenial, and visual cortex and the rest of the brain. The seed locations were primarily chosen for their anatomical distribution rather than a presumed functional role. Seed coordinates were guided by the Paxinos and Franklin mouse atlas and an atlas overlay adapted by White et al. Seed-to-seed connection matrices, global connectivity maps, and interhemispheric homotopic connectivity maps and indices were calculated as described previously. 43 The connection matrix data were also organized as topological circle plots. [00163] Blood pressure, blood gas and CBF measurement: Blood pressures (BPs) were measured via femoral artery catheter (MacLab; ADInstruments, Colorado Springs, MO) under general anesthesia. During the procedure, mice were anesthetized under isoflurane 3% induction and 1-1.5% maintenance in 70% N2O and 30% O2. Mice temperature was maintained at 37°C using rectal temperature and a heating pad. The left femoral artery was catheterized with heparinized PE10 tubing. After cannulation, the animals were waiting for stabilize BPs for minutes. After stabilization, arterial pressure (mmHg) was recorded (PowerLab, ADInstruments, Colorado Springs, CO, USA), and an arterial blood samples were drawn to measure pH, partial pressure oxygen (pO2), partial pressure of carbon dioxide (pCO2) at the same time that diffuse correlation spectroscopy (DCS) measurements were done. Systolic BPs (SBPs), means BPs (MBPs) and dilated BPs (DBPs) were assessed using stabilized value (Buckley et al., J Cereb Blood Flow Metab.2015 Dec; 35(12): 1995–2000; f.hubspotusercontent10.net/hubfs/40716/Resource%20Center%20H ubDB% 20Files/Mouse%20Femoral%20Pressure%20Measurement%20(SP-3-sp) .pdf). [00164] Behavior: AnyMaze software (ver.8.42, Stoelting, Wood Dale, IL, USA) was used for tracking and analysis. For the Open field testing (OFT), mice were placed in a 28ௗ×ௗ18ௗcm open field. Distance traveled and speed were recorded for 30ௗmin. For the Y- maze, we used a Y-shaped apparatus consisting of three 33-cm arms with 15-cm-high walls, and a 7.6ௗ×ௗ7.6ௗ×ௗ7.6-cm triangular intersection. Each arm was identified with a symbol (square, circle, star). Mice were allowed access to the three arms for a total of 5 min, and their movements were recorded by AnyMaze. The number of times the mouse entered all three arms without re-entering the previous arm (i.e. triplets of ABC, ACB, BCA, etc. vs. ABA, CBC, etc.) and the total number of arm entries were recorded. From this, a percent alternation was calculated. The apparatus was cleaned with 70% ethanol between trials. For the rotarod, five trials were performed each day for three days. Mice were placed on a rod at a starting rotation of 4 r/min which was constantly accelerated to 40 r/min over the course of 120ௗs. The latency of falling off of the rod and the rod RPMs were recorded. [00165] Carotid Artery Ligations: Carotid ligations were performed in 10-week-old mice. Briefly, animals were anesthetized using intraperitoneal ketamine/xylazine (80 and 12 mg/kg, respectively) followed by a small incision in the neck to expose the carotid artery. Then the left carotid was ligated at the carotid bifurcation level using an 8-0 silk suture. At 21 days mice were sacrificed and carotids were collected for histological analysis. Approximately 98% of wild-type mice developed stenotic lesions. Mice that developed thrombosis of the ligated carotid were excluded from the study (~1%–2%). All the above mice and wild-type mice (C57BL/6J) were purchased from The Jackson Laboratory. For tissue analysis, animals were euthanized through inhalational isoflurane (Sigma-Aldrich) prior to tissue collection. All carotid ligation (left carotid) procedures were performed at 10 weeks of age. All experiments were performed on male and female animals at a 1:1 ratio. [00166] Carotid Histology. Left and right carotids were cryosectioned using a standard OCT protocol and sectioning. The right carotid artery was unligated and served as an internal control. Neointimal analysis was performed from H&E-stained horizontal cross sections embedded in OCT compound. Briefly, the distal 0.2 mm of carotid from the ligation suture site was discarded followed by generation of 20 slides (10 ^m). Slides 1, 5, 10, and 15 were H&E stained for quantification of neointima. For quantification of the neointima, internal and external elastic lamina perimeters and medial thickness from 4 quadrants were measured and averaged using ImageJ software (NIH). Slides 2–4, 6–9, and 11–14 were used for immunofluorescence staining. Plasmid constructs [00167] Brief descriptions of plasmids can be found in Table 3. Oligonucleotide sequences for amplicon sequencing and digital droplet PCR (ddPCR) can be found in Table 4. All modifications to plasmids were generated through standard molecular cloning via restriction digest and ligation or isothermal assembly. SpCas9 nuclease human expression plasmids were generated by subcloning the open reading frames of different Cas enzymes into RTW3027 (pCMV-T7-SpCas9-BPNLS-3xFLAG-P2A-EGFP) 8 . Adenine base editor (ABE) variants were generated by modifying ABEmax (Addgene plasmid 112101) 23 to alter the Cas coding sequence or to modify the deaminase to the recently described ABE8.20m or ABE8e domains. 15,16 Plasmids encoding the split base editors for packaging in dual-AAV vector constructs were generated by modifying Cbh-v5-AAV-ABE-N-term (Addgene 137177) and Cbh-v5-AAV-ABE-C-term (Addgene 137178). 19 Briefly the N-terminal plasmid was modified to substitute the ABE7.10 domain for ABE8e, and an S55R mutation was added to the SpCas9 coding sequence; the C-terminal plasmid was modified to include the SpCas9-VRQR mutations (D1135V/G1218R/R1335Q/T1337R) and the gRNA spacer sequence targeting the ACTA2R179H allele in the mouse genome (see Table 3). Human cell expression plasmids for U6 promoter-driven SpCas9 sgRNAs were generated by annealing and ligating duplexed oligonucleotides corresponding to spacer sequences into BsmBI- digested BPK1520; 4 U6 promoter-driven SaCas9 sgRNAs were generated by annealing and ligating duplexed oligo nucleotides corresponding to spacer sequences into BsmBI-digested BPK2660 (Kleinstiver et al., Nature Biotechnology, 2015; PMID 26524662). U6 promoter- driven Cas12a crRNAs were generated by annealing and ligating duplexed oligo nucleotides corresponding to spacer sequences into BsmBI-digested BPK3079 and BPK3082 for AsCas12a and LbCas12a, respectively (Kleinstiver et al., Nature Biotechnology, 2019; PMID 30742127). U6 promoter driven pegRNAs were generated by annealing and ligating duplexed oligonucleotides corresponding to the spacer, the sgRNA scaffold, and the RTT/PBS sequence into BsmBI-digested MNW320 (pUC19-U6-[BsmBI]-terminator). Table 3. List of plasmids
Table 4. Oligonucleotides
Human cell culture and transfections [00168] Human HEK 293T cells (ATCC) were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FBS (HI-FBS) and 1% penicillin/streptomycin. All tissue culture reagents were purchased from ThermoFisher. The supernatant media from cell cultures was analyzed monthly for the presence of mycoplasma using MycoAlert PLUS (Lonza). [00169] For HEK 293T cell experiments, ~2x10 4 HEK 293T cells were seeded per well in 96-well plates between 20 and 24 hours prior to transfection. For nuclease experiments, 29 ng of nuclease and 12.5 ng of sgRNA expression plasmids (unless otherwise indicated) were mixed with 0.3 μL of TransIT-X2 (Mirus) in a total volume of 15 μL Opti- MEM (Thermo Fisher Scientific), incubated for 15 minutes at room temperature, and added to HEK 293T cells. For base editor experiments, 70 ng of base-editor and 30 ng of sgRNA expression plasmids were mixed with 0.72 μL of TransIT-X2 in a total volume of 15 μL Opti-MEM, incubated for 15 minutes at room temperature, and added to HEK 293T cells. For prime editor experiments, 70 ng of base-editor, 30 ng of pegRNA, and 30 ng of nicking sgRNA expression plasmids were mixed with 0.93 μL of TransIT-X2 in a total volume of 15 μL Opti-MEM, incubated for 15 minutes at room temperature, and added to HEK 293T cells. Transfections were halted after 72 hours. Genomic DNA was collected by discarding the media, resuspending the cells in 100 μL of quick lysis buffer (20 mM Hepes pH 7.5, 100 mM KCl, 5 mM MgCl2, 5% glycerol, 25 mM DTT, 0.1% Triton X-100, and 60 ng/ul Proteinase K (NEB)), heating the lysate for 6 minutes at 65 ºC then 2 minutes at 98 ºC, and then storing at -20 ºC. Extraction of genomic DNA from mouse tissues [00170] Genomic DNA was extracted from mouse tissues using the Agencourt DNAdvance protocol (Beckman Coulter). Briefly, approximately 10 to 20 mg samples of frozen tissue were incubated at 37 °C for 30 min prior to treatment. Lysis reactions were performed using LBH lysis buffer, 1 M DTT, and proteinase K (40mg/mL) in 200 μL reactions, incubated overnight (18 to 20 hr) at 55 °C with shaking at 100 RPM. Genomic DNA was purified from lysate using Bind BBE solution containing magnetic beads and performing three washes with 70% ethanol. DNA was eluted in 200 μL of Elution buffer EBA, and the approximate concentrations of genomic DNA were quantified by Nanodrop. Next-generation sequencing and data analysis [00171] The genome modification efficiencies of nucleases, base editors, and prime editors were determined by next-generation sequencing using a 2-step PCR-based Illumina library construction method. Briefly, genomic loci were amplified from approximately 100 ng of genomic DNA using Q5 High-fidelity DNA Polymerase (NEB) and the primers listed in Table 4. PCR products were purified using paramagnetic beads prepared as previously described. 7,24 Approximately 20 ng of purified PCR product was used as template for a second PCR to add Illumina barcodes and adapter sequences using Q5 and the primers described in Walton et al., Science, 2020. 8 PCR products were purified prior to quantification via capillary electrophoresis (Qiagen QIAxcel), normalization, and pooling. Final libraries were quantified by qPCR (Illumina Library qPCR Quantification Kit, KAPA Biosystems) and sequenced on a MiSeq sequencer using a 300-cycle v2 kit (Illumina). On-target genome editing activities were determined from sequencing data using CRISPResso2. 25 Levels of editing from the in vivo experiments were calculated as: ( [%CGT in the treated samples] - [%CGT in the control] ) / [%CAT in the control]. Quantification of AAV genomes in mouse tissues [00172] ddPCR reactions to quantify the approximate number of AAV genomes in transduced tissues were performed using approximately 20 to 60 ng of genomic DNA, 250 nM of each primer and 900 nM probe (see Table 4), and ddPCR supermix for probes (no dUTP) (BioRad) in 20 μL reactions. Droplets were generated using a QX200 Automated Droplet Generator (BioRad). Thermal cycling conditions were: 1 cycle of (95 °C for 10 min), 40 cycles of (94 °C for 30 sec, 58 °C for 1 min), 1 cycle of (98 °C for 10 min), hold at 4 °C. PCR products were analyzed using a QX200 Droplet Reader (BioRad) and absolute quantification of inserts was determined using QuantaSoft (v1.7.4). Quantification of Cas9 mRNA [00173] Total RNA was extracted from homogenized liver and Aorta using with TRI Reagent (Ambion) according to the manufacturer's instructions. RNA quality and quantity were assessed using nanodrop spectrophotometer (A260/280 ratio). High-Capacity cDNA Reverse Transcription Kit (thermofisher) was used for quantitative conversion of up to 1 μg of total RNA to single-stranded cDNA. Cas9 gene-specific primer sets (oCA106-oCA109; Table 4) were designed and real-time RT-PCR (qPCR) reactions were performed in a LightCycler (Roche) using SYBR Green to monitor cDNA amplification. Transcript levels of genes were normalized to the expression values of the actin gene. Two technical replicates were done for each combination of cDNA and primer pair, and the quality of the PCR reactions was determined by analysis of the dissociation and amplification curves. [00174] In separate experiments to quantify mRNA expression of either the N- or C- terminal SpCas9 fragments, RNA was extracted using the RNeasy Plus Universal Kit (Qiagen, Hilden, Germany). RNA was reverse transcribed using the RT2 First Strand Kit (Qiagen, Hilden, Germany) and used in ddPCR reactions as described above for gDNA. For ddPCR reactions, cDNA was normalized to 2 ng/μL and each ddPCR reaction contained 12 ng of cDNA, 250 nM of each primer and 900 nM probe (see Table 4), and ddPCR supermix for probes (no dUTP) (BioRad). Cas9 Enzyme Immunofluorescence [00175] Liver tissue was fixed in 4% PFA and embedded in OCT following by cryosectioning at 8mm. Then antigen retrieval was performed on a pressure cooker using the Borg Decloaker buffer (Biocare medical, Cat# BD1000G1). Liver sections were blocked with donkey serum at 10% for 1 hour followed by incubation overnight at 4C with anti-mouse Cas9 (Cell signaling, Cat# 14697S) at 1:100 dilution. Slides were washed with PBS-tween at 0.1% 3 times for 3 min followed by incubation with secondary antibody (1:400) (ThermoFisher Scientific, USA) for 1hr at room temperature. Then slides were washed with PBS-tween at 0.1%, 4 times for 3 min each and slides were mounted with diamond mounting medium containing DAPI. Slides were visualized with the Leica TCS SP8 confocal microscopy station and micrographs were digitized with the Leica Application Suite X software. Example 1. Neurovascular Dysfunction and Neurodegeneration in a Mouse Model of Multisystemic Smooth Muscle Dysfunction Syndrome [00176] To characterize the neurovascular and systemic features of MSMDS we created a knock in mouse expressing a mutant SMA with a missense mutation at position 179 replacing arginine with histidine (Acta2 R179fl/+ ) under the control of tissue-specific cre excision. Herein, we characterize the multisystemic features of the animal model and an extensive and neurovascular evaluation to gain insight into the human syndrome. [00177] Creation of ACTA2 R179fl/+ knock in mouse - To characterize the neurovascular features of MSMDS a Cre-inducible ACTA2 R179H knock in mouse was created. The mouse contains a mutant exon 6 downstream of a polyA terminator and surrounded by loxP sites. Cre mediated excision advances the mutant exon to be spliced into the ACTA2 mRNA allele, resulting in expression of a mutant alpha smooth muscle actin within cell types expressing a Cre recombinase (Fig.1A). To activate the R179H allele in multiple smooth muscle containing organs we used cre recombinase driven by a smooth muscle cell myosin heavy chain cre (Myh11-cre) and to activate the allele within the anterior cerebral circulation without other systemic effects, we used the neural crest specific cre allele (Wnt1-cre) 20 . Systemically, Myh11-Cre:Acta2 R179fi/+ mice were smaller, gained weight slower, and had early mortality when compared to either Wnt1-Cre:Acta2 R179fl/+ or Acta2 R179fl/+ littermates. (Fig.1B-C). Other phenotypes reminiscent of MSMDS were notable including abdominal distention with thinning of the abdominal wall suggestive of Prune-Belly syndrome (Fig.1C) and a primary pulmonary emphysema, both of which have been seen in human patients. Fig. 1D is a picture of the abdominal wall of a control and a Myh11-Cre:Acta2R179Hfl/+ mice, illustrating that the Myh11-Cre:Acta2R179Hfl/+ is thin and commonly exhibits clearly visible distension. Mice are 4 months of age at the time of the picture. Gross examination of abdominal organs demonstrates striking abnormalities including dilated bowel, hydronephrosis, and distended bladder (Fig.1F-H). Bladder dysfunction was evident through analysis of voiding patterns in Myh11-Cre:Acta2 R179fi/+ mice. Necropsy of spontaneous death in Mhy11-Cre:Acta2 R179Hfl/+ mice revealed no evidence of aortic rupture but frequently necrotic, distended bowel loops. [00178] Characterization of smooth muscle cells in ACTA2 R179fl/+ knock in mouse – We cultured vascular smooth muscle cells from the aortas of Myh11-Cre:Acta2 R179fl/+ and control mice to determine cellular level changes in phenotype. Smooth muscle cells from Myh11-Cre:Acta2 R179fl/+ mice were noted to adopt a less flattened cellular shape in comparison to wild type Acta2 R179fl/+ control cells, although filopodial structures were maintained resulting in a more spindle-like appearance (Fig.2A). The Myh11-Cre allele expressed a green fluorescent protein, allowing for verification of cellular identity. To examine the differences in cellular morphology we probed the structure of the filamentous actin cytoskeleton with rhodamine-labeled phalloidin. We also probed for the localization of SMA, the smooth muscle myosin (MYH11), and the actin cytoskeleton during stress fiber formation through the application of transforming growth factor beta (TGF-beta) (Fig.2B). As previously described the ligand, TGF-beta, stimulated the formation of numerous thickened stress fibers in wild type Acta2 R179fl/+ control cells, while Myh11-Cre:Acta2 R179fl/+ cells could not form stress fibers (Park et al., Biochem Biophys Res Commun.2017 Jan 29;483(1):129-134). At lower resolution, SMA staining often appeared diffuse and not arranged in filamentous structures in Myh11-Cre:Acta2 R179fl/+ VSMCs. In fact, higher resolution magnification of the filamentous cytoskeleton in these cells also revealed both a lack of colocalization of SMA with F-actin and underdevelopment of stress fibers in Myh11- Cre:Acta2 R179fl/+ cells (Fig.2C). [00179] Cardiovascular features of ACTA2 R179fl/+ knock in mouse - Conduit arterial failure is a common feature of MSMDS in human patients. The hearts of Myh11- Cre:ACTA2 R179fli/+ and Wnt1-Cre:ACTA2 R179fl/+ mice were normal in appearance (not shown), however there were multiple abnormalities of the aortas and carotid arteries of the mutant mice. Diameters of the ascending aorta and the proximal right brachiocephalic artery were significantly larger at two months of age in both Myh11-Cre:ACTA2 R179fli/+ and Wnt1- Cre:ACTA2 R179fl/+ mice when compared to wild type ACTA2 R179fl/+ littermates via echocardiography or gross examination (Fig.3A-B). Aortas from Myh11-Cre:ACTA2 R179fli/+ and Wnt1-Cre:ACTA2 R179fl/+ mice exhibited cellular disarray, a thickened wall, and increased collagen deposition in the adventitia (Fig.3C). Immunofluorescence demonstrated characteristics similar to other models of vascular dysfunction, prominently including loss of filamentous actin staining in the vascular smooth muscle cells (VSMCs) (Fig.3D). The contractile marker SM22a was depleted in diseased aortic tissue, while staining for both MYH11 and SMA itself were substantively unaffected (Fig.3D). Physiological assessments of Myh11-Cre:Acta2R179Hfl/+ mutant mouse showed a trend towards lower systolic and pulse blood pressures when compared to controls (Fig.3E). [00180] Neurobehavioral Features of the ACTA2 R179H knock in mouse – Patients with MSMDS suffer from fatigue, exercise intolerance and impairment of behavioral/cognitive ability (anxiety, executive and attention deficits). Up to a third of patients will motor disability (cerebral palsy). 18 To assess whether our animal model demonstrates similar signs of this clinical disease we performed a series of behavioral assessments. Open field test (OFT) and Y-maze testing showed decreased distance traveled and average walking speed at baseline at 12, 16, 20 and 24 weeks of age in Wnt1- Cre:Acta2 R179Hfl/+ mice compared to littermate controls (Fig.4A-B). Furthermore, we found increased anxiety as evidenced by increased thigmotaxis in the Wnt1-Cre:Acta2 R179Hfl/+ mouse (Fig.4A). Y-maze testing suggests similar working spatial memory on the percent alternation assessment. However, the Wnt1-Cre:Acta2 R179Hfl/+ mice had fewer entries into each arm of the Y-maze which suggests a lower level of motor activity, consistent with open field testing (Fig.4B). There was a significant decrease in the latency of falling in rotarod testing in the Acta2 R179Hfl/+ mice (Fig.4C) without evidence of motor asymmetries or coordination issues as measured by the modified neurological severity scores (mNSS) 5 which includes beam walk test. [00181] Neuropathologic and Cerebrovascular Features of the ACTA2 R179H knock in mouse – To seek pathologic correlates of these behavioral abnormalities we next examined the cerebral and cerebrovascular systems of the ACTA2 R179H knock in mouse. Wnt1-Cre:Acta2 R179Hfl/+ mice showed narrowing of the intracranial ICA relative to its extracranial portion and pathognomonic straightening of its branching after latex injection (Fig.5A). Histologic evaluation of intracranial arteries demonstrated disrupted elastin fibers, increased collagen deposition in the vessel wall, narrowing of the vessel lumen, and significant wall thickening. These features were seen earlier in the Myh11-Cre:Acta2 R179Hfl/+ mice at 8 weeks, (Fig.5B-G) compared to 16 weeks in the Wnt1-Cre:Acta2 R179Hfl/+ (not shown). Cerebellar vessels were not affected in the Wnt1-Cre:Acta2 R179Hfl/+ , as expected given lack of Cre recombination in VSMCs in the vertebrobasilar system (derived from somatic mesoderm). At the parenchymal level both Wnt1-Cre:Acta2 R179Hfl/+ and Myh11- Cre:Acta2 R179Hfl/+ mice showed signs of ischemic neurodegeneration affecting both grey and white matter structures with an earlier and more severe phenotype in the Myh11- Cre:Acta2 R179Hfl/+ mice. Significant white matter degeneration with loss of volume, vacuolation and enlarged peri-vascular spaces were seen in the corpus callosum, anterior commissure and internal capsule (plus cerebellar white matter in Myh11- Cre:Acta2 R179Hfl/+ mice) (Fig.6A-B). Cortical neurons with degenerative features – including red neurons, ghost neurons and neurons with swollen nuclei and intracytoplasmic neuronal inclusions akin neurofibrillary tangle - were observed in the motor/somatosensory cortices, caudoputamen and hippocampus (Fig.6C). Quantification of dentate gyrus neurons in the hippocampus showed >50% count loss at 10 weeks of life (Fig.6D). No significant difference in these indices were observed in a heterozygous Acta2 +/- mouse when compared to WT suggesting that a single copy of the gene is sufficient to prevent the cerebrovascular phenotype (data not shown). [00182] Neurovascular Connectivity - Neuronal activity creates a hemodynamic response that locally alters brain concentrations of deoxy- and oxy-hemoglobin producing a time-dependent signal representation of CBV that can be assessed by non-invasive functional optical intrinsic signal (OIS) imaging. This imaging can be performed over the dorsal surface of the mouse brain in vivo through chronic windows made by implanting a glass coverslip over an intact and unaltered mouse skull, as previously described 22 (Fig.7A). Analysis of the Wnt1-Cre:Acta2 R179Hfl/+ mice at from 12 to 24 weeks of age showed significant alterations in neural connectivity (NC) affecting a measure of global resting state functional connectivity. Using Avertin anesthesia (which best preserves the hemodynamic response 23 ) we observed lower amplitude CBV fluctuations in the BOLD frequency range (0.01-0.1 Hz) early in the Wnt1-Cre:Acta2 R179Hfl/+ mice while Acta2 R179Hfl/+ controls reached these levels 3 months later (Fig.7B). Acta2 R179H mice had a significant decrease in global and motor areas connectivity at first assessment followed by worsening and loss of connectivity in sensory, retrosplenial, and visual areas by 6 months of age (Fig.7C). The severe and early motor connectivity loss and neurodegeneration found on histological studies of the Acta2 R179Hfl/+ mice may explain our behavioral findings. Notably, loss of functional connectivity in the retrosplenial cortex, which heaviest projections originate in the hippocampal formation, predicts cognitive aging. 24 [00183] Induced Stroke in ACTA2 R179H mice - Mice expressing the ACTA2 R179H allele in the cerebral vasculature exhibit vascular abnormalities, ischemic neurodegeneration and subsequent connectivity and behavioral abnormalities. To simulate the hypoperfusion event- related ischemic injury observed in patients with MSMDS we induced cerebral hypoperfusion through unilateral surgical carotid arterial occlusion. In wild type mice, unilateral carotid artery occlusion is well tolerated, necessitating the creation of bilateral carotid artery occlusion models to achieve significant ischemic cerebral damage. 25,26 In contrast, in our Wnt1-Cre:Acta2 R179Hfl/+ and Myh11-Cre:Acta2 R179Hfl/+ mouse models of disease, we noted significant mortality after unilateral carotid artery ligation, in contrast to control mice (Acta2 R179Hfl/+ ). To determine if this mortality was due to ischemic injury/stroke, we used histology to examine the brains of mice subjected to unilateral carotid artery occlusion (Fig.8A). Immunofluorescent staining for hypoxia inducible factor 1-alpha (HIF1a), a marker of tissue ischemia, demonstrated strong signal in the Wnt1- Cre:Acta2 R179Hfl/+ mice, at three days post ligation, accentuated on the side ipsilateral to the carotid ligation, but no significant HIF1a staining on either side of the brain in the ligated Acta2 R179Hfl/+ littermate (Fig.8A). H&E and Trichrome staining clearly showed evidence of ischemic tissue damage in the ipsilateral Wnt1-Cre:Acta2 R179Hfl/+ mouse neural tissue (Fig 8A, top row). Examination of carotid artery histology in the Myh11-Cre:Acta2 R179Hfl/+ mice after carotid artery occlusion, revealed excessive neointimal proliferation in the ligated artery when compared to Acta2 R179Hfl/+ mice (Fig.8B), a finding noted in Acta2 -/- mice. Additionally, we noted necrosis of tissue supplied by the external carotid artery including the eye (cornea, conjunctiva) and the external auricle ipsilateral to common carotid occlusion highly suggestive of lack of ability to engage collateral blood flow from the contralateral external carotid via common anastomosis, another sing of SMC vessel dysfunction and lack of flow redistribution capacity (Fig.8C). Example 2. Altered actin and cytoskeleton in patients with ACTA2 R179 mutations. [00184] In this experiment, human skin fibroblasts were grown from a patient with an ACTA2 R179H mutation, a patient with ACTA2 R179C mutation, and a control (unaffected) subjects. Cell contents were fractionated through centrifugation to separate polymerized filamentous beta actin (F-actin) from globular beta actin (G-actin). Beta actin was identified via western blot with antibody directed against it (Cytoskeleton Inc., Clone 7A8.2.1). The results, shown in Fig.9, demonstrate that cells expressing the mutant ACTA2 proteins have a decreased amount of polymerized (F-actin) and altered ratios of G/F actin in the affected subjects. [00185] In addition, the same cells were stained for the presence of polymerized beta- actin (F-actin) with phalloidin-FITC and an Į-smooth muscle actin antibody, and the cell nuclei were stained with DAPI. To induce a smooth muscle-like state the cells were incubated in the presence of TGF-beta (10ng/mL) for 24 hrs. Stress fiber induction was less robust in R179H and R179C patient cells. In addition, in human cells with R179H or R179C ACTA2 mutations, Į-smooth muscle actin failed to colocalize efficiently to stress fibers, as seen in wild type cells. [00186] A scratch assay was also performed. In this experiment human skin fibroblasts were grown from a patient with an ACTA2 R179H mutation, a patient with ACTA2 R179C mutation, and two control (unaffected) subjects. The cells were plated on cell chambers that contained an insert of consistent width that prevented cellular growth in that area. The insert was removed, and the cells were photographed at time zero and at 24 hours later. Cells then migrated into the space previously occupied by the insert. The results, seen in Fig.10A, showed that cells from patients with R179H and R179C migrate into the space faster than control cells. TGF-beta treatment was found to slow the migration of both control and patient cells (see Fig.10B). Example 3. Minimal HDAC9 Promoter Directs Protein Expression in Dysfunctional VSMCs [00187] To investigate the feasibility of using the human HDAC9 promoter in gene therapeutic applications within VSMCs, we first examined the expression of the native Hdac9 protein within the murine aorta in an experimental model of multisystemic smooth muscle dysfunction syndrome (MSMDS). As compared to Acta2 R179Hfl/+ mice (wild type), aortas from Myh11-Cre:Acta2 R179Hf/+ (disease) mice demonstrated upregulation of the Hdac9 protein by immunoblotting, coincident with downregulation of alpha smooth muscle actin (ACTA2) and calponin (CNN1)(Fig.11A). The promoter region of HDAC9 contains multiple identifiable trans-acting sites including sites for KLF factors, STAT proteins, NFAT factors, as well as MEF2 binding sites as previously described 40 (Fig.11B). To find a smaller portion of the --2500 to +0 bp, herein named P1, promoter region with disease-induced upregulation activity we created smaller fragments (-2500-1300 bp, P2) and (-1300 to +0 bp, P3) to investigate individually (Fig.11C). We fused each of these fragments to a pcDNA3- RFP reporter for in vitro expression in cells. The individual plasmids were transfected into two types of cells: skin fibroblasts isolated from patients with MSMDS (ACTA2 R179H mutation) or control fibroblasts as well as control human VSMCs to which we added the actin depolymerizing agent, latrunculin. In both cases, we noted induced expression of the HDAC9 promoter either comparing wild type fibroblasts to diseased (ACTA2 R179H mutation) fibroblasts, or in wild type VSMCs upon the application of latrunculin (Figs.11D- F). Also, in both cell types the highest expression was seen with the P2 fragment driving the expression of the RFP reporter. [00188] To determine the suitability of the P2 HDAC9 promoter fragment to direct disease-induced expression of proteins within VSMCs we created an Adeno-associated virus, serotype 8 (AAV8) that directs expression of RFP under the control of the P2 HDAC9 promoter fragment (AAV8-P2-RFP) (Figs.12A-D). After transfection of 1x10^11 vp/kg into either Acta2 R179Hfl/+ (control), or Myh11-Cre:Acta2 R179Hlf+ (MSMDS) mice we evaluated multiple tissues for expression of RFP. As shown by colocalization with Į-SMA, AAV8-P2- RFP directed RFP expression in the vascular compartment of the brain (Fig.12A), aorta (Fig. 12B), and kidney (Fig.12C) in the Myh11-Cre:Acta2 R179Hfl (MSMDS) but not Acta2 R179Hfl/+ (control) mice. Expression in liver, which is highly transduced by AAV8 vectors (rational for serotype selection in this experiment), and its vasculature (Fig.12D) was not enriched overall demonstrating improved VSCM selectivity and lower off-target liver transduction with this promoter gene therapy approach. [00189] Finally, we tested the ability of the P2 HDAC9 promoter fragment to transactivate transcription in multiple vascular disease models including Myh11- Cre:Acta2 R179Hfl (MSMDS), Fbn1 C1039G/+ (Marfan syndrome) 41 , Col3a1 +/- :Col5a +/- (a model of coronary artery dissection, SCAD), and CMV-Cre:Smad4 I499Vfl/+ (Myhre syndrome). In these models we noted the expression of the RFP construct in vasculature and SMC of multiple organs including brain, heart, kidney, and bladder (Figs.13A-D). These data indicate that the HDAC9 P2 promoter fragment has general applicability to other vascular disease entities. Example 4. Generating ACTA2-R179H cell lines [00190] Here we investigated the potential of CRISPR-Cas editors to model and correct a substitution of arginine positioned at residue 179 for a histidine, in the ACTA2 gene, due to a single base missense mutation (CGTĺCAT), referred to herein as the ACTA2 R179H mutation (Fig.14a). Generation of cell lines [00191] Cell lines containing the human ACTA2 R179H mutation were generated by transfecting HEK 293T cells with a prime editor expression plasmid encoding an EGFP reporter (pCMV-PE2-P2A-EGFP; HES1117) along with pegRNA and PE3 nicking sgRNA expression plasmids (HES902 and HES724, respectively). HES902 has a spacer sequence of GCCCCATGCCATCATGCGTC (SEQ ID NO:80), and a pegRNA sequence of GCCAGATCCAGATGCATGATGGCA (SEQ ID NO:81); HES724 has a spacer sequence of GGCCTCACCAGTAGTAACGA (SEQ ID NO:82). Approximately 72 hours post transfection, EGFP positive cells were sorted into a 96-well plates, grown for two passages, and then dilution plated into several 96-well plates. Individual clones were monitored by microscopy and genotypes were analyzed by extracting genomic DNA, performing PCR and sending for Sanger sequencing, or performing PCR and performing amplicon sequencing (as described below). A second round of clone dilution and selection was also performed to enrich for more stable single cell genotypes. Clones harboring ACTA2 R179H genotypes were expanded, and aliquots of ~3x10 6 cells were frozen in complete media with 10% extra FBS for storage in LiN2. Characterization of ACTA2-R179H cell lines [00192] To evaluate safe and effective genome editing approaches to correct the ACTA2 R179H mutation, we first developed cell lines bearing the R179H substitution. We generated ACTA2 R179H mutation in HEK 293T cells, owing to their ease of transfection and culturing, which would permit high-throughput evaluation of dozens of CRISPR-Cas enzymes, base editors, and gRNA combinations. To generate the R179H cell line, we designed and tested three approaches: (1) nuclease-based homology-directed repair (HDR) using a single-stranded DNA donor (ssODN) template encoding the R179H mutation, (2) base editing to convert Arg179 to His, and (3) prime editing by various combinations of PE and pegRNA constructs (Fig.14b). With the HDR approach, we achieved efficient R179H generation but that was also accompanies by high levels of insertion or deletion mutations (indels) in the population of cells, making it unlikely to identify a homozygous R179H or heterozygous R179(wild-type)/R179H clone. With base editing targeting the non-coding strand, depending on the gRNA and CBE constructs we observed R179H editing but also observed bystander edits of nearby bottom strand cytosines (which would additionally generate unwanted M178I or D181K mutations). Finally, by selecting the most optimal permutations of PE and pegRNA combinations for prime editing, we achieved ~12% ACTA2 R179H in bulk cell transfections. [00193] Amongst the HDR and prime editing approaches to generate ACTA2 R179H lines, we cloned out three cell lines and genotyped them. The approximate genotypes of these cell lines, where the ACTA2 locus in HEK 293T cells appears to be triploid, were R179/R179/R179H (line 1), R179H/R179H/indel(15 bp deletion) (line 2), and R179/R179H/indel(5bp insertion) (line 3) (Figs.14C-14E, respectively). Given the genotypes of these cell lines, we then dilution plated these cell lines in an attempt to get purer R179 or R179H allele balances. In doing so, we were able to derive four additional lines with cleaner genotypes, including R179H/indel(15 bp deletion) (line 4), and R179H/R179H (homozygous mutant) (line 5). Subsequent molecular characterization of ACTA2 protein expression patterns using western blot to quantify levels of globular (G) and polymerized (F) actin showed decreased amount of polymerized (F-actin) and higher G/F ratios than wild type HEK cells in mutant cells (Figs.14F-G). Together, these cell lines offer a range of R179H genotypes to test various corrective genome editing strategies. Example 5. Assessment of A-to-G base editing approaches to correct ACTA2 R179H [00194] We initially selected one cell line bearing a heterozygous R179H mutation (line 1, Fig.14C) to use as a model for examining approaches based on ABE-mediated sequence correction (Fig.15A). To determine whether we could use ABEs to revert the disease-causing G-to-A change (CGT>CAT), we designed various gRNAs that would position the edit window of the ABE deaminase domain over the mutated nucleotide (relying on the WT SpCas9 that can target sites with NGG PAMs, the SpG variant for targeting sites with NGN PAMs 8 , the SpCas9-VRQR variant for targeting sites with NGA PAMs 4,6 , and the SaCas9-KKH variant for targeting sites with NNGRRT PAMs 5 (see Figs.15B-E, respectively). These target sites would position the target adenine for the R179H change in either the 8 th , 7 th , 4 th , or 5 th position of the spacer sequence, respectively (see Figs.15B-E). We also explored different deaminase components of the ABE, testing ABE7.10, ABE8.20m, and ABE8e domains 10,15,16 (which exhibit varied A-to-G base editing efficiencies). Excitingly, next-generation sequencing (NGS) results from transfections using these ABEs and gRNAs in the ACTA2 R179H cell line 1 revealed that several enzymes could efficiently reduce the proportion of R179H (CAT) alleles in the population of cells (Fig.15F). Focusing only on the R179H allele, many ABEs and gRNAs led to high levels of reversion to the wild- type R179 sequence (Fig.15G). The selection of specific gRNAs, Cas variants, and deaminase domains had a major impact on the editing efficiency. These results establish the feasibility of using base editors to directly correct the ACTA2 R179H mutation. There are additional combinations of gRNAs (e.g., new target sites or varying the spacer length), Cas enzymes, and ABE domains that can be explored to improve editing potency and selectivity (Table 1). [00195] Next we performed additional base editing experiments using a second cell line bearing the R179H/R179H/indel genotype (line 2, Fig.14D). We selected a subset of the best-preforming base editors from our initial experiment and transfected them into R179H line 2. Once again, we observed very robust levels of R179H correction (Fig.15H). However, this time we also analyzed the edit purity, detecting also editing of an adenine upstream that can cause an M178V mutation. By plotting the fraction of perfect H179R corrections, perfect H179R corrections with the M179V bystander edit, and M179V bystander edits only, our data revealed that the SpG and VRQR variants with the ABE8e or ABE8.20 domains and using the HES1208 gRNA (with an NGA PAM) offer the highest levels of ‘pure’ H179R correction (Fig.15H). Example 6. Allele-specific editing approaches to knockout the ACTA2 R179H allele [00196] Further, we explored an allele-selective knockout approach to selectively destroy the R179H allele (Fig.16A). Since the ACTA2 R179H variant is a dominant negative gain of function mutation, we hypothesized that knockout of the R179H allele without creating indels on the wild-type R179 allele might also be a viable therapeutic strategy. We selected the heterozygous R179H cell line (line 1, Fig.14C) as a model for examining allele- selective editing since it harbors both wild-type and R179H sequences. Following transfection of the Cas nucleases and gRNAs into R179H line 1, three days later we extracted genomic DNA and performed targeted NGS to assess the levels of remaining R179H allele. Our sequencing revealed that several Cas enzymes are capable of knocking down the proportion of the R179H allele (Fig.16B), with wild-type SpCas9 exhibiting the greatest levels of R179H allele reduction. When programmed with two of the gRNAs that we assayed, wild-type SpCas9 selectively knocked-out between 17.6-43.0% of the mutant R179H allele (Fig.16C). There are additional combinations of gRNAs (e.g. new target sites or varying the spacer length) and Cas nucleases that can be explored to improve editing potency and selectivity (Table 2). Example 7. An engineered peptide displaying AAV9 capsid, AAV-PR, mediates a vasculature-enriched transduction phenotype in brain after intravenous delivery. [00197] We packaged a single stranded AAV-CBA-Cre genome into capsids comprising the peptide, PRPPSTH (SEQ ID NO:1), named AAV-PR, and injected this vector (1x10 12 vg/mouse) into the tail vein of adult Ai9 mice that have a Cre-sensitive CAG-floxed- STOP-tdTomato reporter in all cells. Any cells that were successfully transduced by the AAV vector and have Cre-expressed should result in tdTomato expression. Mice were sacrificed three weeks post injection, brains sectioned, and tdTomato detected with immunofluorescence staining. In mice injected with AAV-PR, we observed a distinct vasculature immunostaining of tdTomato expression throughout the entire brain (Figs.17A- D). This profile is in stark contrast to the tropism of parental AAV9-CBA-Cre in adult Ai9 mice, which mediates transduction of mostly astrocytes and neurons. Example 8. Expression of Base editors and CRISPR targeting ACTA2 in SMCs in vitro and in vivo [00198] Given the promise of our base editor results, which demonstrated the feasibility of reverting the ACTA2 R179H CAT codon back to the canonical CGT codon, we explored the use of additional engineered ABEs in hope of achieving higher levels of correction. To do so, we utilized our homozygous HEK 293T ACTA2 R179H cell line to perform additional transfections. We assessed the editing efficiencies of ABE8e 16 versions of WT SpCas9, SpCas9-VRQR 4,6 (harboring D1135V/G1218R/R1335Q/T1337R mutations), SpCas9-VRQR harboring an activity-enhancing S55R mutation 17 , SpG 8 , and two PAM- relaxed variants SpCas9-NRRH and SpCas9-NRCH 18 . We tested these enzymes using all or some of the sgRNAs targeting the three sites harboring NGA, NGC, or NGG PAMs that would appropriately place the target adenine in position A4, A7, or A8 of the edit window of the base editor (Fig.18A). Approximately 72 hours post-transfection, genomic DNA was harvested, and we performed amplicon sequencing to analyze editing in the ACTA2 gene at or near codon R179 (Fig.18A). Similar to our previous results, we observed high levels of intended CAT-to-CGT editing in the target ACTA R179H codon with ABE8e-SpCas9 using the A8 gRNA (Fig.18A. With ABE8e-SpCas9-VRQR and ABE8e-SpCas9-VRQR(S55R), we observed high levels of editing only with the A4 gRNA, consistent with the preference of this SpCas9 variant to recognize sites with NGA PAMs. Given prior nuclease data showing the superiority of the SpCas9-VRQR(S55R) over SpCas9-VRQR 17 , we observed a substantial improvement in A-to-G editing with ABE8e-SpCas9-VRQR(S55R) (Fig.18A). Next, with ABE8e-SpG we observed modest levels of editing with any of the three sgRNAs (since all target sites harbor NGG PAMs), with ABE8e-SpCas9-NRRH we observed low levels of editing with the A4 NGA PAM gRNA, and with ABE8e-SpCas9-NRCH we observed high levels of editing with the A7 NGC PAM gRNA (Fig.18A). However, closer inspection of the mutation profile across each allele revealed that many of the high efficiency ABE8e-gRNA combinations led to bystander edits of unknown consequences (Fig.18B). In fact, only ABE8e-SpCas9-VRQR, ABE8e-SpCas9-VRQR(S55R), and ABE8e-SpG when paired with the A4 NGA PAM gRNA resulted in ‘clean’ edits of the H179R reversion only. Use of ABE8e-SpCas9 or ABE8e-SpG with the A8 NGG PAM gRNA, and ABE8e-SpG or ABE8e-SpCas9-NRCH with the A7 NGC PAM gRNA led to dual H179R and M178V editing (Fig.18B); we observed co-occurrence of H179R and D181 edits in a small fraction of reads ABE8e-SpCas9-VRQR, ABE8e-SpCas9-VRQR(S55R), and ABE8e-SpG when paired with the A4 NGA PAM gRNA. From these results, we proceeded with ABE8e- SpCas9-VRQR(S55R) and the A4 NGA PAM gRNA for in vivo mouse studies, given that we observed the most efficient editing with this construct and also the purest edit outcomes for H179R only. Evaluation of split base editors for packaging into AAV vectors [00199] Thus far our assessment of editing efficiency had been performed using a typical two-plasmid method, where one plasmid encodes the ABE and a second encodes the gRNA (Fig.19a). Towards assessment of in vivo editing, we therefore generated plasmids encoding these constructs in AAV vectors with the goal of producing AAVs. Due to the large sizes of SpCas9-based base editor coding sequences, we endeavored to split the ABE8e- SpCas9-VRQR(S55R) sequence into two parts, similar as to what has been done before for other ABE7.10 and CBE constructs. The first AAV construct encodes the ABE8e domain and an N-terminal portion of SpCas9 fused to the N-terminal end of the split alpha subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) 20–22 (Fig. 19b). The second AAV construct encodes the C-terminal Npu intein domain fused to the C- terminal domain of SpCas9 along with the SpCas9 gRNA expression cassette (Fig.19b). Notably, the N-terminal SpCas9 construct includes the D10A nickase mutation (required for efficient base editing) and the activity-enhancing S55R mutation 17 , while the C-terminal SpCas9 construct encodes the VRQR mutations. We performed transfections in our homozygous ACTA2 R179H HEK 293T cell line to compare ABE8e-SpCas9-VRQR to ABE8e-SpCas9-VRQR(S55R) with P2A-EGFP or P2A-mScarlet reporters all in single plasmid architectures with a separate A4 NGA PAM gRNA plasmid (Fig.19a), along with the dual-AAV split ABE8e-SpCas9-VRQR(S55R) architecture encoding the A4 NGA PAM gRNA in the second AAV construct (Fig.19b). We observed comparable editing between each condition, suggesting that the dual AAV plasmid architecture could lead to high levels of correction of the R179H mutation (Fig.19c). In vivo editing of mouse ACTA2 R179H (Myh11-Cre:Acta2R179Hfl/+ genotype) [00200] Encouraged by these findings, we applied base editing in vivo to correct our mouse model of ACTA2 R179H (C57BL/6 Myh11-Cre:Acta2R179Hfl/+ mutant mice) which as shown herein recapitulates hallmark symptoms of the human brain, aorta and multisystemic pathological phenotypes. The Acta2R179Hfl allele contains 2 copies of exon 6 (Fig.1A), one with a CGT codon (Wild type) and one with a CAT codon (mutant) at position 179, complicating interpretation of sequence based analysis of editing efficiency. After cre- mediated excision the wild type codon within the Acta2R179Hfl allele is excised (CGT codon). Therefore within Cre-expressing cells within the mouse, the Acta2R179Hfl allele only contains the mutant codon (CAT codon). Since it is useful to compare editing efficiency in animals that have an activated allele and are therefore phenotypically affected, this necessitates an unequal codon frequency in experimental animal tissues when compared to control tissues. The baseline codon frequency in the Myh11-Cre:Acta2R179Hfl/+ mutant animals is a 1:1 ratio of the wild-type allele (one CGT codon and one CAT codon) within smooth muscle cells. In the same Myh11-Cre:Acta2R179Hfl/+ mutant animals, cells not expressing cre recombinase have a codon ratio of 2:1 (one CAT codon and two CGT codons). Similarly, in control animals with the genotype Acta2R179Hfl/+ , all cells contain the 2:1 codon ratio (one CAT codon and two CGT codons). To account for this complexity, we designed sequencing primers that do not recognize the altered wildtype codon in the Acta2R179Hfl allele making sequencing interpretable as a baseline 1:1 CAT to CGT codon ratio within cre-expressing cells and 1:1 CGT to CGT codon ratio in cells that do not express cre recombinase (see below). [00201] Using the above mentioned dual-AAV split ABE8e-SpCas9-VRQR(S55R) packaged into two different AAV viral vectors (AAV9 and our novel AAV-PR) we injected Acta2R179Hfl/+ control and Myh11-Cre:Acta2R179Hfl/+ mutant mice. To compare the effects of injection route and timing on in vivo editing we performed retro-orbital injection of 2-week (2-3-week-old mice) (n = 9) and 6-week-old mice (n = 7). Two week-old mice were injected with 1.3e13 vg/kg of each AAV for a total of 2.7e13 vg/kg AAV per mouse (1.6e11 vg/6grs mouse total AAV) and 6-week-old mice were injected with 4e13 vg/kg of each AAV vector for a total injection of 8e13 vg/kg AAV per mouse (1.2e12gc/15grs mouse total AAV). Mice were euthanized at the age of six weeks and editing and Cas9 enzyme mRNA and protein expression were evaluated in various tissues following standard DNA, RNA and protein extraction protocols (see Fig.20A-D). [00202] Extracted genomic DNA from both cohorts of the Acta2R179Hfl/+ control and Myh11-Cre:Acta2R179Hfl/+ mutant mice treated with AAV9, AAV-PR, or untreated was used to analyze editing at the Acta2 R179H locus, by performing amplicon sequencing. Irrespective of the actual genotype and cre recombination status of the animals in these injections, our sequencing approach amplifies a basal genotype of a 1:1 ratio of the wild-type allele (R179 with CGT codon) and mutant allele (R179H with CAT codon) given that our primers do not recognize the altered sequence of the wild-type exon 6 in the mutant allele before or after cre recombination. Thus, our primers only amplify the mutant copy on the mutant allele and the wild-type locus of a wild-type allele, and do not amplify or sequence the wild-type exon 6 encoded on the mutant allele. Amplification and sequencing of this locus led us to observe variable levels of editing in various tissues depending on the cohort of the animals and the serotype of AAV used in the injections (Figs.21A-H). Notably, the younger mice of cohort #2 (2-week-old) showed higher transduction and CAT to CGT editing levels in the brain vasculature when the dual-AAV split ABE8e-SpCas9- VRQR(S55R) was delivered with AAV-PR when compared to AAV9. Liver editing was also more efficient in younger mice when using AAV-PR vector despite a relative lower amount of AAV genome copies potentially suggesting higher levels of ABE expression following cell transduction. Untreated mice showed no transduction or editing in any tested tissues (Figs. 21A-H). [00203] To analyze the levels of AAV transduction in each tissue and in each cohort of injections, we utilized the extracted genomic DNA (containing AAV genomes) to perform ddPCR (Figs.22A-G). As expected, for both AAV9 and AAV-PR, we observed the highest levels of AAV transduction in the livers of both cohorts of animals, though this observation was much more pronounced in cohort 1 where direct intravenous injection via tail vein was used and when AAV-PR was used as the vector (Fig.20A). To determine Cas9 enzyme expression in liver and vasculature (aorta) Cas9 mRNA quantification using qPCR and Cas9 immunofluorescence were performed. Higher levels of Cas9 mRNA expression were found in the vasculature (aorta) of 2-week-old mice (cohort #2) treated with dual-AAV-PR when compared to AAV9 (Fig.23A). Moreover, Cas9 immunofluorescence confirmed primarily vascular (arterial, vein and sinusoidal capillaries) Cas9 protein expression in the liver 2 weeks after injections (Fig.23B). The lower expression in hepatocytes maybe due to the high mitotic rates during the liver growth of young mice, diluting the episomal Cas9 expression of subsequent hepatocyte generations. In contrast, the low mitotic rates of vascular cells (endothelium, pericytes and SMCs) contribute to the longer Cas9 expression. [00204] These data taken together show detectable levels of A-to-G editing in both control and mutant mice (both harboring a copy of the mutant allele R179H with CAT codon) that are encouraging particularly in light of the short term survival allotted to these mice. In young mice (cohort #2) a trend towards higher editing efficiency was observed when injected with dual-AAV-PR split ABE8e-SpCas9-VRQR(S55R) compared to the dual-AAV9 split ABE8e-SpCas9-VRQR(S55R) injected group (See Figs.24A and 24B). Example 9. In vivo editing of ACTA2 R179H (C57BL/6 Myh11- Cre:Acta2R179Hfl/+ genotype) mice at p3 – therapeutic efficacy [00205] To determine therapeutic efficacy in vivo we proceeded to injected 3 day-old mice with the above mentioned dual-AAV split ABE8e-SpCas9-VRQR(S55R) packaged into two different AAV viral vectors (AAV9 and our novel AAV-PR) at 1.3e 13 vg/kg of each AAV for a total of 2.7e 13 vg/kg AAV per mouse (1.6e 11 vg/2grs mouse total AAV) into Acta2R179Hfl/+ control and Myh11-Cre:Acta2R179Hfl/+ mutant mice via retro-orbital injection (n = 6 control and 6 mutant mice for AAV-PR and n= 6 controls and 5 mutant for AAV9) (Figs.25A-C). Mice were euthanized at the age of eight weeks and survival, rotarod test, aortic root diameter determined by echocardiography (ECHO), weights, open field test performance, histology, AAV genome biodistribution and A-G editing efficiency were evaluated in various tissues following standard DNA, RNA and protein extraction protocols. Treatment with dual-AAV split ABE8e-SpCas9-VRQR(S55R) editors resulted in increased survival and normalization of weight gain, aortic root diameter, rotarod and open field behavioral performances of Myh11-Cre:Acta2R179Hfl/+ mutant mice (see Figs.26, 27, 28, 29A-B, and 30A). Histological analyses of organs and brain vasculature showed normalization of vessel diameters, tortuosity and smooth muscle cell contractility in muscular arteries demonstrated by wrinkling of interna elastica layer and ACTA2 (SMA) staining is shown in Fig.30B. No severe toxicity from the treatment was observed in all injected mice up to day of sacrifice. [00206] These data taken together demonstrate in vivo that A-G editing leading to a significant prevention of disease phenotype in an animal model of the disease. [00207] Assessment of on-target R179H editing in a cohort of P3 AAV-injected mice [00208] To determine whether injections of the ABE-encoding AAV vectors earlier in the life of the animals may lead to increased correction of R179H, we injected an additional cohort (#3) of our mouse model of ACTA2 R179H (C57BL/6 Myh11-Cre:Acta2R179Hfl/+). We performed P3 injections with AAV9 or AAV-PR packaged dual AAV vectors encoding the split ABE8e-SpCas9-VRQR(S55R) construct with the A4 NGA PAM gRNA via retro- orbital injection using 1.3e 13 vg/kg of each N- and C-terminal vector for a total of 2.6e10 13 vg/kg per mouse (with n = 8 and n = 14 mice for AAV9 and AAV-PR, respectively). Mice were euthanized after 8 weeks and genomic DNA was extracted from sections of various tissues. [00209] We analyzed editing at the humanized Acta2 R179H locus by amplicon sequencing in both Acta2R179Hfl/+ control and Myh11-Cre:Acta2R179Hfl/+ mutant mice. A-to-G editing to correct R179H was assessed in bulk tissue across all treatment groups without selection or sorting for transduced cells (Table 5). Consistent with the previous results demonstrating that injections earlier might lead to more efficient correction, we observed substantially higher correction of R179H when injecting at P3 compared to later injections in prior cohorts. When analyzing tissues from mice treated with dual AAV9 vectors encoding ABE8e-SpCas9-VRQR(S55R) with the A4 gRNA, in liver we observed up to 60% A-to-G editing at R179H>R (converting CAT to CGT) versus control samples (Fig. 31A). Across various other tissues including brain vasculature, ascending aorta, heart, lungs, kidney, bladder, small intestine, and large intestine (Figs.31B-I, respectively; Table 5), in many cases we detected appreciable levels of editing in bulk tissue (up to ~3% in brain vasculature and ~10% in heart, without sorting for transduced cells). We then analyzed editing in animals from the same cohort but treated with dual AAV-PR vectors encoding the same ABE and gRNA. In most cases, we observed higher levels of editing from AAV-PR treated mice compared to the AAV9 across nearly all tissues analyzed (Figs.32A-I and Table 5). For instance, we achieved higher average editing in liver reaching ~43.3% with AAV-PR versus ~27.7% mean editing with AAV9 (Fig.31A vs.32A). Similarly, we observed ~4.9% and ~1.2% mean editing in brain vasculature (Fig.31B vs.32B for AAV9 and AAV-PR, respectively) and ~12.1% and ~5.5% mean editing in heart (Fig.31D vs.32D for AAV9 and AAV-PR, respectively). [00210] Together, these results reveal that our in vivo ABE8e-SpCas9-VRQR(S55R) with gRNA A4 editing approach resulted in high levels of ACTA2-R179H correction, especially when the AAV-encoded ABE constructs are delivered early in life. Analysis of unintended edits [00211] We also explored the degree to which this ABE strategy could create different types of unwanted edits at the on-target site when using AAV-delivered ABE8e-SpCas9- VRQR(S55R) with gRNA A4. We analyzed the level of bystander A-to-G editing of two nearby adenine bases that can cause ACTA2 M178V or D181G mutations (located in positions A-1 and A10, respectively, compared to the intended A4 edit; Fig.33; Table 5). Since ABEs are known to also cause unwanted edits at cytosines located in position 6 (C6) of an ABE target site (when the cytosine is found in a thymine-cytosine (TC) sequence context; Kim et al., Nature Biotechnology, 2019, PMID: 31548727; Li et al., Nature Communications, 2020, PMID: 33203850; Jeong et al., Nature Biotechnology, 2021, PMID: 34211162), we also analyzed the propensity for our ABEs to generate bystander C6-to-A, C6-to-G, or C6-to- T edits, which cause amino acid changes L180M, L180V, or a silent L180L, respectively (Fig.33B; Table 5). [00212] For A-1 bystander editing that causes ACTA2 M178V, for mice treated with dual AAV9 vectors, we observed a range of M178V bystander editing across each tissue (Figs.34A-I; Table 5). In liver we observed up to 2% M178V editing (Fig.34A), with lower levels of bystander editing in brain vasculature (<0.4%; Fig.34B), aorta (<0.1%; Fig.34C), or heart (<0.4%; Fig.34D). For most other tissues, we observed levels of bystander editing closer to the levels observed in untreated controls (Figs.34E-I). For mice treated with dual AAV-PR vectors (Figs.35A-I; Table 5), the trend of bystander editing stratified by tissue was similar to what we observed with AAV9 (Fig.34A-I). However, the general levels of bystander editing per tissue with AAV-PR vectors (Figs.35A-I) were higher than what we observed in tissues treated with AAV9 (Figs.34A-I), consistent with our observation of higher levels of intended editing of the A4 base with AAV-PR. [00213] Next, we also analyzed A10 bystander editing that causes ACTA2 D181G (Fig.33). For mice treated with dual AAV9 vectors, we observed a range of D181G bystander editing across each tissue (Figs.36A-I; Table 5). In liver we observed up to 10% D181G editing (Fig.36A), with lower levels of bystander editing in brain vasculature (<1%; Fig.36B), aorta (<0.1%; Fig.36C), or heart (<1.5%; Fig.36D). For most other tissues, we observed levels of bystander editing closer to the levels observed in untreated controls (Figs. 36E-I). For mice treated with dual AAV-PR vectors (Figs.37A-I; Table 5), the trend of bystander editing stratified by tissue was similar to what we observed with AAV9 (Fig.36A- I). However, the mean levels of bystander editing per tissue with AAV-PR vectors (Figs. 38A-I) were higher than what we observed in tissues treated with AAV9 (Figs.37A-I), consistent with our observation of higher levels of intended editing of the A4 base with AAV-PR. [00214] We also analyzed C6A, C6G, or C6T editing that would cause L180M, L180V, or a silent L180L, respectively (Fig.33). For mice treated with dual AAV9 vectors, we observed a range of L180 bystander editing across each tissue (Figs.38A-I; Table 5). In liver we observed up to 3.6% total L180 editing (Fig.38A), with lower levels of bystander editing in brain vasculature (<0.5%; Fig.38B), aorta (<0.12%; Fig.38C), or heart (<0.6%; Fig.38D). For most other tissues, we observed levels of bystander editing closer to the levels observed in untreated controls (Figs.38E-I). For mice treated with dual AAV-PR vectors (Figs.39A-I; Table 5), the trend of bystander L180 editing stratified by tissue was similar to what we observed with AAV9 (Fig.38A-I), though, once again, the mean levels of bystander editing per tissue with AAV-PR vectors were higher, consistent with our observation of higher levels of intended editing of the A4 base with AAV-PR. In general, the most prevalent C6 bystander edit was a C-to-T, which is a silent L180L substitution that we therefore anticipate should not impact the function of ACTA2. Furthermore, the second most common C6 substitution is a C-to-G that causes L180V, leading to a biochemically similar amino acid side chain (similar size and biophysical properties), which may lead to similar ACTA function. The proportion of total C6 bystander edits across each of the three classes of substitutions (C-to-A, C-to-G, and C-to-T) appeared to vary slightly by tissue, consistent with prior reports in different cell lines (Burnett et al., Frontiers in Genome Editing, 2022, PMID: 35910415). [00215] We then analyzed the propensity of our ABE approach to create unwanted insertion or deletion mutations (indels) at the A4 target site (Table 5). For AAV9-treated mice (Figs.40A-I), we sometimes observed indels in the 0.1-1% range for liver, brain vasculature, aorta, or heart (Figs.40A-D, respectively). For the other tissues, indels were typically < 0.05% and in most cases near control levels (Figs.40E-I). In mice treated with AAV-PR vectors (Figs.41A-I), as expected due to higher levels of overall editing, we observed slightly elevated levels of indels compared to AAV9-treated mice (Figs.40A-I and Figs.41A-I, for AAV9 and AAV-PR, respectively). [00216] Despite these collected levels of unwanted editing (bystander editing and/or indels), we observed much higher levels of on-target editing at ACTA2-R179H that led to dramatic phenotypic recovery in the Myh11-Cre:Acta2R179Hfl/+ mice. These observations suggest that the unwanted edits were either innocuous or have little functional consequence (the bystander edits), or occurred at levels that did not detract from the phenotypic recovery of the animals or do not impact their general viability. [00217] AAV transduction and transgene expression [00218] Next, we analyzed the levels of AAV transduction in selected tissues and animals from cohort 3. We initially quantified the approximate number of AAV genomes per haploid genome by performing ddPCR on extracted genomic DNA (containing AAV genomes) from both AAV-PR and AAV9 treated animals (Figs.42A and 42B, respectively). Samples treated with AAV9 appeared to exhibit slightly higher levels of AAV transduction in liver and brain compared to those treated with AAV-PR, with comparable transduction in the heart (Figs.42A and 42B). We then analyzed expression of either the N- or C-terminal mRNA from either AAV by extracting RNA, generating cDNA, and performing ddPCR. Depending on the tissue and AAV serotype, we generally observed comparable levels of expression from the N- and C-terminal AAV constructs (Figs.42C-F). Differences in AAV genome copy number or mRNA expression levels compared to A-to-G editing may be due to cell division and dilution of the AAV genome/transgene across the growth and lifespan of the animals. Assessment of on-target R179H editing in a cohort of P14 AAV-injected mice [00219] We also injected a smaller cohort of mice at P14 using AAV9 packaged dual AAV vectors encoding the split ABE8e-SpCas9-VRQR(S55R) construct with the A4 NGA PAM gRNA, as described above but at a later point in life (P14 instead of P3) (Table 5). Relative to our P3 injections, we observed lower levels of overall editing across various tissues and mice in this small cohort injected later in life (Figs.43A-J). Reduced on-target editing at A4 in this cohort was accompanied by lower overall levels of bystander editing of M178V, D181G, or L180M/V/L editing (Figs.44A-J, 45A-J, and 46A-J, respectively), along with reduced levels of indels (Figs.47A-J). Summary [00220] These additional data from Cohorts 3 demonstrate that injecting the mice with the AAV-encoded ABE and gRNA earlier in life can lead to much higher levels of correction of the ACTA2 R179H mutation, and that we can achieve high levels of intended reversion of ACTA2 R179H to R179 with low levels of unwanted bystander edits or indels. The striking phenotypic recovery that was observed in the mice indicates that the bystander edits (in addition to the R179H correction) might be functionally innocuous or tolerated by the ACTA2 protein (including the biochemically similar L180V substitution or the silent L180L change). TABLE 5. Sequencing results from mouse studies _ _ y yyy yyyyyyy y
* Sample not available LrgInt, Large intestine; SmaInt, Small intestine; Blad, Bladder; BrVasc, Brain vasculature; AsAort, Ascending aorta; DeAort, Descending aorta; Uninject, Uninjected animals. Example 10. Generation and testing of HDAC9 mini promoters [00221] The HDAC9 fragment 2 (P2) identified above containing ~1200 bp was further divided into 3 new fragments of about ~330bp (P2.1, P2.2 and P2.3; FIG.50). Mini promoters were subcloned into a DNA plasmid to drive the expression of the RFP as an indicator of promoter activity.2ug each of plasmids containing P2.1, P2.2 and P2.3 driving expression of red fluorescent protein (RFP) were transfected in wild-type HEK cells.72 hrs. post transfection, cells were fixed and analyzed on a fluorescent microscope. Results show promoter activity for P2.1 and P2.2. as observed by the red color intensity (FIG.51). P2.3 showed lower activity. Western blotting confirmed these results (FIGs.52A-B). REFERENCES 1. Jinek, M. et al. 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