Editing of AATD-related genes using a NME2 base editor

Abstract

Aspects of the disclosure relate to compositions and methods for base editing using viral vectors. In some aspects, the disclosure provides an adenine base editor (ABE) utilizing an evolved Cas9 nickase derived from Neisseria meningitidis; and adeno-associated viruses (AAVs) comprising (or encoding) such adenine base editors. In some aspects, compositions described herein are useful for treatment of certain diseases, for example pulmonary diseases.

Description

[0001] EDITING OF AATD-RELATED GENES USING A NME2 BASE EDITOR

[0002] RELATED APPLICATIONS

[0003] This Application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. provisional application serial numbers 63 / 722,675, filed November 20, 2024, entitled “EDITING OF AATD-RELATED GENES USING A NME2 BASE EDITOR”, and 63 / 641,472, filed May 2, 2024, entitled “EDITING OF AATD-RELATED GENES USING A NME2 BASE EDITOR”. The entire contents of each referenced application are incorporated by reference herein.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The content of the electronic sequence listing (U012070207WO00-SEQ-KZM.xml; Size: 55,054 bytes; and Date of Creation: April 24, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND OF INVENTION

[0007] Alpha- 1 antitrypsin deficiency (AATD) is an inherited monogenic disorder that can lead to lung disease and liver damage. AATD is caused by mutations in the SERPINA1 gene, which encodes AAT — a serine protease inhibitor that is made by hepatocytes and secreted into serum for delivery to the lungs, where it inhibits neutrophil elastase. The most common diseasecausing SERPINA1 variant, known as the “Z” allele (PI*Z), is a G-to-A mutation that causes a glutamic acid to lysine amino acid change at position 342 of AAT. The Z-AAT protein misfolds, polymerizes, and accumulates in the liver, increasing the risk of liver fibrosis. The inability of hepatocytes to secrete polymerized Z-AAT leads to a serum AAT deficiency that causes lung emphysema.

[0008] SUMMARY OF INVENTION

[0009] Adenine and cytidine base editors (ABE or CBE) deaminate a target nucleotide within a defined editing window, defined by a guide RNA (e.g., sgRNA) binding site. ABE deaminates adenine to inosine, which is read as a guanosine during DNA replication or repair, thereby converting A to G. Thus, ABEs are good candidates for Z allele correction. However, some ABEs deaminate not only the target adenine but also “bystander” adenines in the designated editing window, leading to mutations of unknown importance.

[0010] The inventors of the present disclosure have developed a compact ABE utilizing an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2-C). eNme2-C is -20% smaller than SpyCas9, allowing co-delivery of eNme2-C ABE and sgRNA in a single AAV. Moreover, eNme2Cas9 is less promiscuous than SpyCas9, leading to fewer off-target editing events. The inventors have shown that AAV-mediated delivery of eNme2-C ABE can efficiently correct PI*Z in hepatocytes and rescue liver disease in a PI*Z transgenic mouse model.

[0011] Accordingly, in some aspects, the disclosure provides an isolated nucleic acid comprising a transgene flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), the transgene comprising a first nucleic acid encoding an adenine deaminase; a second nucleic acid encoding an RNA-guided nuclease; and a third nucleic acid encoding a guide RNA (gRNA) that comprises a region of complementarity to a nucleic acid encoding a mutant alpha 1 antitrypsin (AAT) peptide.

[0012] In some embodiments, an adenine deaminase is a TadA-8e peptide. In some embodiments, a TadA-8e peptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0013] In some embodiments, an adenine deaminase is a TadA-9e peptide. In some embodiments, a TadA-9e peptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0014] In some embodiments, an RNA-guided nuclease is a Neisseria meningitidis evolved Cas9 nickase (eNme2.C) peptide. In some embodiments, an eNme2.C peptide comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0015] In some embodiments, a gRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 4-6.

[0016] In some embodiments, a transgene further comprises one or more promoters. In some embodiments, a transgene comprises a first promoter and a second promoter. In some embodiments, a first promoter comprises a U1 a promoter. In some embodiments, a first promoter (e.g., Ula promoter) is operably linked to a nucleic acid sequence encoding an adenine deaminase. In some embodiments, a second promoter comprises a U6 promoter. In some embodiments, a second promoter (e.g., U6 promoter) is operably linked to a nucleic acid sequence encoding one or more guide RNAs (gRNAs).

[0017] In some embodiments, a transgene further comprises a linker molecule positioned between a nucleic acid encoding an adenine deaminase and a nucleic acid encoding a RNA- guided nuclease (e.g., an eNme2.C peptide). In some embodiments, a linker molecule comprises an amino acid linker.

[0018] In some embodiments, a mutant alpha 1 antitrypsin (AAT) peptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 7.

[0019] In some embodiments, AAV ITRs are AAV2 ITRs.

[0020] In some aspects, the disclosure provides an isolated nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 8 or 9. In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising an isolated nucleic acid comprising a transgene flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), the transgene comprising a first nucleic acid encoding an adenine deaminase; a second nucleic acid encoding an RNA-guided nuclease; and a third nucleic acid encoding a guide RNA (gRNA) that comprises a region of complementarity to a nucleic acid encoding a mutant alpha 1 antitrypsin (AAT) peptide; and one or more AAV capsid proteins.

[0021] In some embodiments, one or more AAV capsid proteins are AAV8 capsid proteins.

[0022] In some aspects, the disclosure provides a method for reducing a mutant alpha 1 antitrypsin (AAT) peptide level in a subject in need thereof, the method comprising administering an isolated nucleic acid or the rAAV as described herein, to a subject comprising an alpha 1 antitrypsin (AAT) gene comprising one or more mutations.

[0023] In some aspects, the disclosure provides a method for base editing a nucleic acid encoding a mutant alpha 1 antitrypsin (AAT) peptide, the method comprising contacting a cell comprising the nucleic acid with an isolated nucleic acid or the rAAV as described herein.

[0024] In some aspects, the disclosure provides a method for treating a subject having alpha 1 antitrypsin (AAT) deficiency, the method comprising administering to a subject in need thereof an isolated nucleic acid or the rAAV as described herein.

[0025] In some embodiments, one or more mutations comprise a G-to-A mutation that causes a glutamic acid to lysine amino acid change at position 342 of AAT.

[0026] In some embodiments, a subject is a mammal. In some embodiments, a subject is a human.

[0027] BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG. 1A is a figure showing the downstream effects caused by the Z allele (G-to-A mutation that causes a glutamic acid to lysine amino acid change at position 342 of AAT). Sequence shown corresponds to SEQ ID NO: 17.

[0029] FIG. IB is a figure showing the correction of the Z allele by an adenine base editor (reversion of the AAG codon back to GAG codon). Sequences shown correspond to SEQ ID NOs: 17 (top) and 18 (bottom).

[0030] FIG. 2 provides a schematic of an exemplary rAAV comprising a compact ABE utilizing an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2-C) and experiments following administration of said rAAV to a mouse model subject (e.g., a disease model) to determine editing efficiency and disease progression. FIG. 3A provides guide sequences and constructs shown with PI*Z mutation circled and PAM sequence underlined. Sequences shown correspond to SEQ ID NOs: 19 (guide 1) and 20 (guide 2).

[0031] FIG. 3B provides a summary of guides 1 and 2 editing efficiency in HEK293T PiZ reporter cells. Number represents mean editing rate measured with amplicon deep sequencing (n=3). Sequence shown corresponds to SEQ ID NO: 21.

[0032] FIG. 3C provides a graph showing the editing efficiency of an exemplary rAAV comprising eNme2-C ABE8e. Data represents mean with SEM (n=3).

[0033] FIG. 4A provides a graph showing the editing efficiency of an exemplary rAAV comprising eNme2-C ABE9e. Data represents mean with SEM (n=2).

[0034] FIG. 4B provides a graph showing the ratio of bystander / on-target editing for ABE8e versus ABE9e. Analysis was done by dividing editing at each adenine position by A9 editing. Data represents mean with SEM (n=2 for ABE9e, n=3 for ABE8e).

[0035] FIG. 5A provides data from a deep sequencing data showing editing efficiency in mice treated for 4 or 8 weeks. Number represents mean editing rate (n=4 for 4- week timepoint, n=5 for 8 -week timepoint).

[0036] FIG. 5B provides a graph showing the allele percentage in PI*Z mice treated with ABE9e. Line represents median (n=4 for 4-week timepoint, n=5 for 8-week timepoint). Sequences shown correspond to SEQ ID NOs: 22 (top) and 23 (bottom).

[0037] FIG. 5C provides a representative image of PAS-D-stained liver section of untreated or treated mice. White circle shows PAS-D- area. Number of PAS-D- cells in a 20x field is also shown with the line representing median (n=3).

[0038] FIGs. 6A-6F show representative data for optimization of an all-in-one editor for AAT correction. FIG. 6A shows a PiZ mutation site with the pathogenic A mutation circled. Amino acids are in bold. Sequence shown corresponds to SEQ ID NO: 26. FIG. 6B shows two guides were tested for and delivered to PiZ reporter cells. PAM and editing window for each guide are shown. Sequence shown corresponds to SEQ ID NO: 24. FIG. 6C shows A to G editing efficiency of the two guides in PiZ reporter cells. Numbers represent mean, n=3. Target A is noted as A9, with potential bystander edits noted as A3, A7, A10, A12, A13, Au, Ais, A22, and A23. Sequence shown corresponds to SEQ ID NO: 5. FIG. 6D shows editing efficiency of Nme2 base editors with guide 2. Dashed line represents target adenine. Mean ± SD shown, n=3. FIG. 6E shows editing efficiency of all-in-one construct that can deliver eNme2.C-TadA8e. ± SD shown, n=3. FIG. 6F shows mutant- specific sgRNA edits PiZ reporter but not the wildtype PiM with a single G mismatch. Small arrows depict target base while large arrows depict bystanders. Sequence shown corresponds to SEQ ID NO: 5. FIGs. 7A-7D show representative data for reduction of bystander edits with ABE9e. FIG. 7A shows editing efficiency of all-in-one construct that can deliver eNme2.C-TadA9e in reporter cells. Mean± SD shown, n=3. FIG. 7B shows the ratio of bystander to on-target edit in PiZ reporter cells. Mean ± SD shown. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t-test. Sequence shown corresponds to SEQ ID NO: 25. FIG 7C shows allele population of cells edited with eNme2.C-TadA8e or eNme2.C-TadA9e. Fine depicts median, n=3. Sequence shown corresponds to SEQ ID NO: 26. FIG. 7D shows deep sequencing of OT sites. Mean ± SD shown. See Table 1 for additional information, n=2.

[0039] FIGs. 8A-8D show representative data for in vivo editing of PiZ animals. FIG. 8A shows all-in-one eNme2.C-TadA9e AAV design (top) with schematic of tail- vein injection in PiZ animals(bottom). FIG. 8B shows eNme2.C-TadA9e in vivo editing at 4 and 8 weeks in PiZ animals. Numbers represent mean (n=3 for control, n=5 for 4 weeks, and n=7 for 8 weeks). FIG. 8C shows allele percentage in PiZ mice treated with eNme2.C-TadA9e. Fine represents median, n=5 for 4 weeks, n=7 for 8 weeks. Sequence shown corresponds to SEQ ID NO: 26. FIG. 8D shows representative images of mouse liver stained with PAS-D. Mice were either untreated or treated with 9el Ivg AAV for 8 weeks. Scalebar: 500pm for top images, 100pm for bottom images. Squares represent area of zoom from the top images, while circle represents the PASD+ and PAS- regions. Right: quantification. Mean ± SD shown. Significance was calculated by unpaired t-test, n=3.

[0040] FIGs. 9A-9G show representative data for Generation of AAT-null;PiZ animal and lung disease reversal in AAT-null;PiZ animal. FIG. 9A shows a schematic of AAT-null;PiZ mouse generation; a schematic of ABE treatment to study EPS-induced emphysema in AAT-null;PiZ animals; and editing in “AAT-null;PIZ” animals when treated with 9el lvg AAV for 15 weeks (n=4 for control, n=6 for ABE). FIG. 9B shows AAT levels in control and treated AAT-null;PiZ animals. Mean ± SD shown, n=3 for control, n=5 for ABE. Both groups were treated with EPS. FIGs. 9C-9G show inspiratory capacity (FIG. 9C), compliance (FIG. 9D), elastance (FIG. 9E), resistance (FIG. 9F) and (FIG. 9G) pres sure- volume (PV) loop of PBS, EPS, or ABE+EPS treated animals (n=7 for PBS group, n=10 for EPS group, and n=9 for ABE+EPS group). Fine depicts median. Significance was detected by one-way ANOVA with uncorrected Fisher’s least significance difference, except for PV-loop (two-way ANOVA with Dunnett. Significance is between PBS and EPS group; ABE+EPS and EPS group. Mean + SEM shown), *p < 0.05,****p < 0.0001.

[0041] FIG. 10 shows editing at the endogenous SERPINA1 gene in the PiZ reporter cell line. Dashed line depicts target base. Sequence shown corresponds to SEQ ID NO: 27. FIG. 11 shows PiZ animals were treated with 8el lvg all-in-one eNme2.C-TadA8e for 8 weeks. Mean ± SD shown, n=3.

[0042] FIG. 12 shows AAT levels in AAT-null;PiZ animals when Z-AAT levels are deducted. Mean ± SD shown, n=5.

[0043] FIG. 13 shows the allele percentage of AAT-null;PiZ animals treated with eNme2.C- TadA9e for 15 weeks. Line represents median, n=6.

[0044] DETAILED DESCRIPTION OF INVENTION

[0045] Alpha- 1 antitrypsin disease (AATD) is an inherited monogenic disorder that can lead to lung disease and liver damage. It is caused by mutations in the SERine Proteinase Inhibitor family A member 1 (SERPINAP) gene, which encodes AAT protein - a serine protease inhibitor. AAT is produced by hepatocytes in the liver and is delivered via serum to lungs, where it inhibits neutrophil elastase. The most common AATD allele - called PI*Z - is a G-to-A mutation that produces a dysfunctional misfolded protein, Z-AAT, that aggregates in hepatocytes, which can cause liver disease; reduced serum AAT causes pulmonary emphysema. Currently, the only approved therapy for AATD emphysema is costly, weekly infusions of purified AAT for life. AATD exhibits a remarkably strong founder effect in Europe and North America. In fact, greater than 95% of patients with severe AATD lung disease possess at least one PiZ allele. This unusual genetic homogeneity of the AATD population makes it uniquely suitable for a base editing strategy.

[0046] Traditional gene therapies are being developed to augment AAT function in patients. Strategies focus on the delivery of adeno-associated virus (AAV) vectors encoding a wild-type SERPINA1 transgene or modulating RNA levels of the mutant transcript. However, these approaches still require repeated treatment or do not reach the therapeutic threshold needed to ameliorate disease phenotype. A one-time treatment that corrects the SERPINA1 gene and halts AATD progression can significantly improve patient quality of life.

[0047] Base editors (BE) have revolutionized gene therapy treatments due to their ability to make precise edits. Currently, the two major classes of BEs are cytosine base editors (CBEs) which make C-to-T edits, and adenine base editors which make A-to-G edits (ABEs). Despite their promise, BEs have been challenging to deliver with a single AAV due to the packaging limit (~4.7kb cargo size) that must encapsulate the BE, guide cassette, promoters, and regulatory elements. The most commonly used Cas9 is from Streptococcus pyogenes (SpCas9), which alone is 1368 amino acids, and hence is unable to be packaged into a single AAV as an ABE system. AAVs have been widely used as a gene therapy tool in clinics, as they have low integration risk, low immunogenicity, and can target a myriad of tissues by switching the virus serotype. However, its packaging limit stands at ~4.7kb. Given that base editors with the most widely used Streptococcus pyogenes Cas9 (SpCas9) are already ~5.2kb without the guide and regulatory sequences, other editors are needed. A single AAV can minimize AAV dosage, thereby reducing potential toxicity and increasing editing efficiency since cells only need a single vector for editing to occur.

[0048] CRISPR / Cas9-mediated homology directed repair (HDR) can correct PI*Z in the liver and partially restore serum AAT levels in an AATD mouse model. Yet, HDR is limited by the need to deliver a DNA repair template, its inefficiency in non- and slow-dividing cells, and its generation of genotoxic double- strand breaks. By contrast, CRIS PR-mediated adenine base editors (ABEs) support precise editing without requiring a DNA donor or double strand breaks. ABE consists of adenine deaminase (TadA) conjugated to Cas9 nickase. When directed by a guide RNA to a specific sequence, ABE deaminates adenine in a defined editing window. The resulting inosine is read as guanosine, thereby converting A to G. Thus, ABE is a good candidate for PI*Z correction.

[0049] Adenine base editor (ABE) consists of an adenine deaminase (TadA) conjugated to a Cas9 nickase, mediating A to G base editing within a defined editing window. While ABEs based on SpCas9 have been used to correct the PI*Z mutation, they were too large for efficient therapeutic delivery via a single adeno-associated virus (AAV) due to the SpCas9 protein size. Provided herein is a compact, evolved Cas9 nickase derived from Neisseria meningitidis (eNme2-C) that can be packaged using a single AAV. The editing efficiency of an AAV comprising a compact, evolved Cas9 nickase derived from Neisseria meningitidis (eNme2-C) may be assessed at the PI*Z site. In some embodiments, not only was there high editing efficiency, but bystander editing rates can be decreased depending on the TadA deaminase used.

[0050] Aspects of the disclosure relate to compositions (e.g., isolated nucleic acids, rAAV vectors, rAAVs, etc.) and methods for gene editing. The disclosure is based, in part, on isolated nucleic acids encoding combinations of gene editing proteins (e.g., Cas proteins) and base editors (e.g., adenosine deaminase proteins) with certain regulatory sequences that are amenable to packaging in recombinant adeno-associated viruses (rAAVs). In some embodiments, isolated nucleic acids and vectors described herein do not exceed the packaging capacity of recombinant adeno-associated virus (rAAV) particles.

[0051] Accordingly, in some aspects, the disclosure provides an isolated nucleic acid comprising a transgene flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), the transgene comprising a first nucleic acid encoding an adenine deaminase; a second nucleic acid encoding an RNA-guided nuclease; and a third nucleic acid encoding a guide RNA (gRNA) that comprises a region of complementarity to a nucleic acid encoding a mutant alpha 1 antitrypsin (AAT) peptide. In some embodiments, compositions of the disclosure are useful for base editing nucleic acid sequences encoding mutant Alpha- 1 antitrypsin (AAT). In some embodiments, compositions of the disclosure are useful for treating Alpha- 1 antitrypsin (AAT) deficiency in a subject in need thereof.

[0052] Alpha-1 Antitrypsin Deficiency

[0053] Alpha- 1 antitrypsin (AAT), also known in the art as serpin peptidase inhibitor, clade A (SERPINA1), is a protein that functions as proteinase (protease) inhibitor. AAT is mainly produced in the liver, but functions in the lungs and liver, primarily. As used herein the term, “alpha- 1 antitrypsin deficiency” refers to a condition resulting from a deficiency of functional AAT in a subject. In some embodiments, a subject having an AAT deficiency produces insufficient amounts of alpha- 1 antitrypsin. In some embodiments, a subject having an AAT deficiency produces a mutant AAT. In some embodiments, insufficient amounts of AAT or expression of mutant AAT results in damage to a subject’s lung and / or liver. In some embodiments, the AAT deficiency leads to emphysema and / or liver disease. Typically, AAT deficiencies result from one or more genetic defects in the AAT gene. The one or more defects may be present in one or more copies (e.g., alleles) of the AAT gene in a subject. Typically, AAT deficiencies are most common among Europeans and North Americans of European descent. However, AAT deficiencies may be found in subjects of other descents as well.

[0054] Subjects (e.g., adult subjects) with severe AAT deficiencies are likely to develop emphysema. Onset of emphysema often occurs before age 40 in human subjects having AAT deficiencies. Smoking can increase the risk of emphysema in subjects having AAT deficiencies. Symptoms of AAT deficiencies include shortness of breath, with and without exertion, and other symptoms commonly associated with chronic obstructive pulmonary disease (COPD). Other symptoms of AAT deficiencies include symptoms of severe liver disease (e.g., cirrhosis), unintentional weight loss, and wheezing. A physical examination may reveal a barrel-shaped chest, wheezing, or decreased breath sounds in a subject who has an AAT deficiency.

[0055] The following exemplary tests may assist with diagnosing a subject as having an AAT deficiency: an alpha- 1 antitrypsin blood test, examination of arterial blood gases, a chest x-ray, a CT scan of the chest, genetic testing, and lung function test. In some cases, a subject having or suspected of having an AAT deficiency is subjected to genetic testing to detect the presence of one or more mutations in the AAT gene. In some embodiments, one or more of the mutations listed in Table 1 are detected in the subject.

[0056] In some cases, a physician may suspect that a subject has an AAT deficiency if the subject has emphysema at an early age (e.g., before the age of 45), emphysema without ever having smoked or without ever having been exposed to toxins, emphysema with a family history of an AAT deficiency, liver disease or hepatitis when no other cause can be found, liver disease or hepatitis and a family history of an AAT deficiency.

[0057] In some embodiments, alpha- 1 antitrypsin deficiency can result in two distinct pathologic states: a lung disease which is primarily due to the loss of anti-protease function, and a liver disease due to a toxic gain of function of the mutant AAT protein (e.g., mutant PiZ- AAT). For example, since mutant AAT-PiZ exhibits a gain-of-function hepatocellular toxicity accumulating in the endoplasmic reticulum, therapies aimed at decreasing AAT-PiZ mRNA levels may ameliorate or even reverse the liver pathology. In addition, increased secretion of functional AAT protein protects the lungs from neutrophil elastase and associated proteolytic enzymes. Applicants have developed several rAAV vectors that provide for delivery of base editors targeted against mutant AAT.

[0058] Isolated Nucleic Acids

[0059] A "nucleic acid" sequence refers to a DNA or RNA sequence. In some embodiments, proteins and nucleic acids of the disclosure are isolated. As used herein, the term “isolated” means artificially produced. As used herein, with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins or peptides, the term “isolated” refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0060] In some embodiments, an isolated nucleic acid comprises a transgene encoding one or more adenine deaminase proteins (also referred to as adenosine deaminase proteins). Adenine deaminases are a group of enzymes that catalyze the conversion of adenine to hypoxanthine and ammonia. In the context of nucleic acids, adenine deaminases form part of the purine degradation pathway. Catalytic activity of adenine deaminases on DNA may result in an “A “to “G” shift via deamination of adenine to inosine, which is read as a “G” during DNA repair processes. In some embodiments, an adenine deaminase is a TadA adenine deaminase. TadA is a tRNA-specific adenosine deaminase derived from bacteria, for example Escherichia coli, as described by Wolf et al. EMBO J., 2002, Jul 15;21( 14):3841-3851. In some embodiments, a TadA adenine deaminase comprises the amino acid sequence set forth in NCBI RefSeq Accession Number WP_001297409.1. In some embodiments, an adenine deaminase protein is a TadA variant, for example a protein having at least 50%, 60%, 70%, 80%, 90% 95%, or 99% identity to a wildtype TadA amino acid sequence (e.g., the amino acid sequence set forth in NCBI RefSeq Accession Number WP_001297409.1). In some embodiments, a TadA variant is a TadA-8e variant, for example as described by Tu et al. Mol Ther. 2022 Jul 12;30(9):2933-2941. In some embodiments, a TadA-8e variant comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a TadA variant is a TadA-9e variant, for example as described by Chen et al. Nature Biotechnology volume 41, pages663-672 (2023). In some embodiments, a TadA-9e variant comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0061] In some embodiments, an isolated nucleic acid as described herein comprises a recombinant gene editing protein, for example an RNA-guided nuclease (RGN). As used herein, the terms “endonuclease” and “nuclease” refer to an enzyme that cleaves a phosphodiester bond or bonds within a polynucleotide chain. Nucleases may be naturally occurring or genetically engineered. Genetically engineered nucleases are particularly useful for genome editing and are classified into four families: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (e.g., engineered meganucleases) and CRISPR-associated proteins (Cas nucleases). Examples of RGNs include but are not limited to Casl3 nucleases, Cas9 nucleases, Cas6 nucleases, Cfpl nucleases, and variants thereof. A variant of a RGN may comprise or consist of a nucleic acid sequence that comprises one or more substitutions, insertions, and / or deletions relative to a wild-type RGN nucleic acid sequence. In some embodiments, an RGN is a catalytically inactive RGN, such as an RGN that retains RNA binding functionality but lacks nuclease activity. In some embodiments, a catalytically inactive RGN is a dead Cas (dCas) nuclease.

[0062] The term “CRISPR” refers to “clustered regularly interspaced short palindromic repeats,” which are DNA loci containing short repetitions of base sequences. CRISPR loci form a portion of a prokaryotic adaptive immune system that confers resistance to foreign genetic material. Each CRISPR loci is flanked by short segments of "spacer DNA", which are derived from viral genomic material. In the Type II CRISPR system, spacer DNA hybridizes to transactivating RNA (tracrRNA) and is processed into CRISPR-RNA (crRNA) and subsequently associates with CRIS PR-associated nucleases (Cas nucleases) to form complexes that recognize and degrade foreign DNA. In certain embodiments, the nuclease is a CRISPR- associated nuclease (Cas nuclease). Examples of CRISPR nucleases include, but are not limited to Cas9, dCas9, Cas6, Cpfl, and variants thereof. In some embodiments, a Cas protein is modified (e.g., genetically engineered) to lack nuclease activity. For example, dead Cas9 (dCas9) protein binds to a target locus but does not cleave said locus. In some embodiments, a Cas protein or variant thereof does not exceed the packaging capacity of a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector, for example as described by Ran et al. (2015) Nature. 520(7546); 186-91. For example, in some embodiments, a nucleic acid encoding a Cas protein is less than about 4.6 kb in length.

[0063] In some embodiments, a nuclease is a variant of a Cas protein. In some embodiments, the nuclease is a variant of a Cas9 protein. In some embodiments, the Cas9 protein variant is derived from a Neisseria meningitidis Cas9 protein. In some embodiments, the Cas9 protein variant is Neisseria meningitidis evolved Cas9 nickase (eNme2.C), for example as described by Huang et al. Nat Biotechnol. 2023 Jan;41(l):96-107. In some embodiments, eNme2.C comprises an amino acid sequence that is at least 60%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the eNme2.C comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0064] For the purpose of genome editing, the CRISPR system can be modified to combine the tracrRNA and crRNA into a single guide RNA (sgRNA), also referred to herein as a “gRNA.” As used herein, the terms “guide RNA,” “gRNA”, and “sgRNA” refer to a polynucleotide sequence that is complementary to a target sequence (e.g., a mutant AAT nucleotide sequence) in a cell and associates with a Cas nuclease, thereby directing the Cas nuclease to the target sequence. In some embodiments, a gRNA (e.g., sgRNA) ranges between 5 and 25 nucleotides in length. In some embodiments, a gRNA (e.g., sgRNA) ranges between 10 and 22 nucleotides in length. In some embodiments, a gRNA (e.g., sgRNA) ranges between 14 and 24 nucleotides in length. In some embodiments, a gRNA (e.g., sgRNA) is longer than 24 nucleotides in length, for example 25, 30, 40, or 50 nucleotides in length. In some embodiments, a Cas protein and a guide RNA (e.g., sgRNA) are expressed from the same vector.

[0065] In some embodiments, a nucleic acid (e.g., gRNA) has a region of complementarity that is perfectly complementary to a portion of a target nucleic acid (e.g., target DNA or target RNA). However, it should be appreciated that in some embodiments, a nucleic acid may be used that has less than 100% sequence complementarity with a target nucleic acid. A nucleic acid (e.g., gRNA) may comprise a region of complementarity that is complementary with sequence as set forth in SEQ ID NO: 7. The region of complementarity of the nucleic acid (e.g., gRNA) may be complementary with at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15 or more consecutive nucleotides of a target nucleic acid (e.g., the sequence as set forth in SEQ ID NO: 7). In addition, to minimize the likelihood of off-target effects, a nucleic acid (e.g., gRNA) may be designed to ensure that it does not have a sequence (e.g., of 5 or more consecutive nucleotides) that is complementary with an off-target nucleic acid.

[0066] Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of a nucleic acid (e.g., gRNA) is capable of hydrogen bonding with a nucleotide at the corresponding position of a target nucleic acid (e.g., target DNA or target RNA), then the nucleic acid and target nucleic acid are considered to be complementary to each other at that position. The nucleic acid (e.g., gRNA) and target nucleic acid are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. Thus, “complementary” is a term that is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the nucleic acid (e.g., gRNA) and target nucleic acid. However, it should be appreciated that 100% complementarity is not required. For example, in some embodiments, a nucleic acid (e.g., gRNA) may be at least 80% complementary to (e.g., at least 85%, 90%, 91%, 92%, 93%, 940%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of a target nucleic acid.

[0067] Thus, it is understood in the art that a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable. In some embodiments, a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable when binding of the sequence to the target nucleic acid produces the desired base editing to occur and there is a sufficient degree of complementarity to avoid non-specific binding to non-target nucleic acids under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.

[0068] Sequence identity, including determination of sequence complementarity for nucleic acid sequences, may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. In some embodiments, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology=# of identical positions / total # of positionsxlOO), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0069] In some embodiments, each region of the transgene can be directly fused to each other, or fused by a polypeptide linker. Polypeptide linkers have been previously described, e.g., in Chen et al., Fusion Protein Linkers: Property, Design and Functionality, Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. In some embodiments, linkers with shorter length are compatible with the isolated nucleic acid described herein because of the packaging size limitation of an AAV vector. In some embodiments, the polypeptide linker is a glycine- serine (GS) linker. In some embodiments, the expression cassette comprises one or more GS linker between the regions fused by linkers.

[0070] In some embodiment, each of the regions of the expression cassette can be placed upstream (e.g., 5’ relative to) or downstream (e.g., 3’ relative to) of each other. In some embodiments, the first region (e.g., the TadA peptide) is upstream of the third region (e.g., the gRNA). In some embodiments, the first region (e.g., the TadA peptide) is downstream of the third region (e.g., the gRNA). In some embodiments, the second region is a Cas protein (e.g., eNME2-C), and is placed downstream of the first region (e.g., TadA peptide). In some embodiments, the second region is a NES, and is placed upstream of the first region (e.g., dCasl3b). In some embodiments, the second region is a Cas protein (e.g., eNME2-C), and is placed upstream of the third region (e.g., gRNA). In some embodiments, the transgene, from 5’ to 3’, comprises a TadA peptide, a linker, a eNME2-C peptide, a linker, and a gRNA.

[0071] In some embodiments, the transgene of the isolated nucleic acid further comprises a promoter operably linked to the first region. In some embodiments, the expression cassette of the isolated nucleic acid further comprises a promoter operably linked to the third region. In some embodiments, the expression cassette of the isolated nucleic acid further comprises a promoter operably linked to the first region and the third region. In some embodiments, the expression cassette of the isolated nucleic acid further comprises a promoter operably linked to the first region, second region, and the third region. In some embodiments, the expression cassette of the isolated nucleic acid further comprises a promoter operably linked to the first region, second region, the third region and fourth region. In some embodiments, the expression cassette comprises one or more nucleic acid sequences operably linked to one or more promoters. The promoters may be the same promoters or different promoters. In some embodiments, two nucleic acid sequences are operably linked to the same promoter. In some embodiments, the promoter is a U6 promoter or a murine small nuclear RNA (Ula) promoter.

[0072] As used herein, a nucleic acid sequence (e.g., coding sequence) and regulatory sequences are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5’ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame.

[0073] A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively linked," "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. In some embodiments, a transgene comprises a nucleic acid sequence encoding a TadA peptide (e.g., a TadA-8e or TadA-9e peptide) operably linked to a first promoter and gRNA encoding sequence operably linked to a second promoter. Generally, a promoter can be a constitutive promoter, inducible promoter, or a tissue- specific promoter.

[0074] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen]. In some embodiments, a promoter is an RNA pol II promoter. In some embodiments, a promoter is an RNA pol III promoter, such as U6 or Hl. In some embodiments, a promoter is an RNA pol II promoter. In some embodiments, a nucleic acid encoding a TadA peptide is operably linked to a Ula promoter. In some embodiments, a nucleic acid sequence encoding a gRNA is operably linked to a RNA pol III promoter. In some embodiments, the RNA pol III promoter is a U6 promoter.

[0075] Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex) -inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547- 5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0076] In another embodiment, the native promoter for the transgene will be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue- specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0077] In some aspects, the disclosure relates to isolated nucleic acids comprising an expression cassette that comprises one or more miRNA binding sites. Without wishing to be bound by any particular theory, incorporation of miRNA binding sites into gene expression constructs allows for regulation of transgene expression (e.g., inhibition of transgene expression) in cells and tissues where the corresponding miRNA is expressed. In some embodiments, incorporation of one or more miRNA binding sites into a transgene allows for de-targeting of transgene expression in a cell-type specific manner. In some embodiments, one or more miRNA binding sites are positioned in a 3’ untranslated region (3’ UTR) of a transgene, for example between the last codon of a nucleic acid sequence encoding a Cas protein or TadA protein, and a poly A sequence.

[0078] In some embodiments, a transgene comprises one or more (e.g., 1, 2, 3, 4, 5, or more) miRNA binding sites that de-target expression of the proteins from central nervous system (CNS) cells. In some embodiments, an expression cassette comprises one or more (e.g., 1, 2, 3, 4, 5, or more) miRNA binding sites that de-target expression of the proteins from immune cells e.g., antigen presenting cells (APCs), such as macrophages, dendrites, etc.). Incorporation of miRNA binding sites for immune-associated miRNAs may de-target transgene expression from antigen presenting cells and thus reduce or eliminate immune responses (cellular and / or humoral) produced in the subject against products of the transgene, for example as described in US 2018 / 0066279, the entire contents of which are incorporated herein by reference.

[0079] The term “orientation” as used herein in connection with transgenes, refers to the directional characteristic of a given expression cassette or structure. In some embodiments, transgene harbors a promoter 5’ of the encoding nucleic acid sequence, and transcription of the encoding nucleic acid sequence runs from the 5’ terminus to the 3’ terminus of the sense strand, making it a directional cassette (e.g., 5’-promoter / (intron) / encoding sequence-3’). Since virtually all expression cassettes are directional in this sense, those of skill in the art can easily determine the orientation of a given expression cassette in relation to a second nucleic acid structure, for example, a second expression cassette, a viral genome, or, if the cassette is comprised in an AAV construct, in relation to an AAV ITR.

[0080] For example, if a given nucleic acid construct comprises two expression cassettes in the configuration 5 ’-promoter 1 / encoding sequence 1— promoter2 / encoding sequence 2-3’,

[0081] »>»»>»»>»»>»»> »>»»»»»»»>»»> the expression cassettes are in the same orientation, the arrows indicate the direction of transcription of each of the cassettes. For another example, if a given nucleic acid construct comprises a sense strand comprising two expression cassettes in the configuration

[0082] 5 ’-promoter 1 / encoding sequence 1— encoding sequence 2 / promoter 2-3’,

[0083] »»»»»>»»>»»»> <<<<<<<<<<<<<<<<<<<<< the expression cassettes are in opposite orientation to each other and, as indicated by the arrows, the direction of transcription of the expression cassettes, are opposed. In this example, the strand shown comprises the antisense strand of promoter 2 and encoding sequence 2.

[0084] For another example, if an expression cassette is comprised in an AAV construct, the cassette can either be in the same orientation as an AAV ITR, or in opposite orientation. AAV ITRs are directional. For example, the 3 TR would be in the same orientation as the promoter 1 / encoding sequence 1 expression cassette of the examples above, but in opposite orientation to the 5TTR, if both ITRs and the expression cassette would be on the same nucleic acid strand.

[0085] A large body of evidence suggests that multicistronic expression constructs often do not achieve optimal expression levels as compared to expression systems containing only one cistron. One of the suggested causes of sub-par expression levels achieved with multicistronic expression constructs comprising two or more promoter elements is the phenomenon of promoter interference (see, e.g., Curtin JA, Dane AP, Swanson A, Alexander IE, Ginn SL. Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Ther. 2008 Mar;15(5):384-90; and Martin-Duque P, Jezzard S, Kaftansis L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2004 0ct;15(10):995-1002; both references incorporated herein by reference for disclosure of promoter interference phenomenon). Various strategies have been suggested to overcome the problem of promoter interference, for example, by producing multicistronic expression constructs comprising only one promoter driving transcription of multiple encoding nucleic acid sequences separated by internal ribosomal entry sites, or by separating cistrons comprising their own promoter with transcriptional insulator elements. All suggested strategies to overcome promoter interference are burdened with their own set of problems, though. For example, singlepromoter driven expression of multiple cistrons usually results in uneven expression levels of the cistrons. Further some promoters cannot efficiently be isolated and isolation elements are not compatible with some gene transfer vectors, for example, some retroviral vectors.

[0086] In some embodiments, a multicistronic expression construct is provided that allows efficient expression of a first encoding nucleic acid sequence driven by a first promoter and of a second encoding nucleic acid sequence driven by a second promoter without the use of transcriptional insulator elements. Various configurations of such multicistronic expression constructs are provided herein, for example, expression constructs harboring a first expression cassette comprising an intron and a second expression cassette positioned within the intron, in either the same or opposite orientation as the first cassette. Other configurations are described in more detail elsewhere herein.

[0087] In some embodiments, multicistronic expression constructs are provided allowing for efficient expression of two or more encoding nucleic acid sequences. In some embodiments, the multicistronic expression construct comprises two expression cassettes. In some embodiments, a first expression cassette of a multicistronic expression construct as provided herein comprises a first RNA polymerase II promoter and a second expression cassette comprises a second RNA polymerase II promoter. In some embodiments, a first expression cassette of a multicistronic expression construct as provided herein comprises an RNA polymerase II promoter and a second expression cassette comprises an RNA polymerase III promoter.

[0088] The isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), or a variant thereof. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, an expression cassette and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The isolated nucleic acid may comprise a region encoding, for example, a protein and / or an expression control sequence (e.g., a poly-A tail), as described elsewhere in the disclosure.

[0089] Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the disclosure is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, an isolated nucleic acid encoding a transgene is flanked by AAV ITRs (e.g., in the orientation 5’-ITR-transgene-ITR-3’). In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, at least one of the AAV ITRs is a AITR, which lacks a terminal resolution site and induces formation of a self-complementary AAV (scAAV) vector. In some embodiments, the AAV ITRs are selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR.

[0090] An expression cassette of an isolated nucleic acid described by the disclosure may further comprises a polyadenylation (poly A) sequence. In some embodiments, a transgene comprises a poly A sequence is a rabbit beta-globulin (RBG) poly A sequence. In some embodiments, a transgene comprises a poly A sequence is a rabbit beta-globulin (RBG) poly A sequence,

[0091] In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 8 or 9.

[0092] In some aspects, the present disclosure also provides a separate isolated nucleic acid encoding one or more guide RNAs to be delivered with (e.g., concurrently or sequentially) with the isolated nucleic acid comprising an expression cassette encoding a first region comprising a TadA peptide ; a second region comprising a catalytically inactive Cas protein (e.g., eNME2-C); and a third region comprising a gRNA that binds to a nucleotide sequence encoding a mutant AAT protein (e.g., SEQ ID NO: 7).

[0093] In some embodiments, an isolated nucleic acids encode one or more gRNAs and may be referred to as a multi-gRNA expression cassette. In some embodiments, a multi-gRNA expression cassette comprises one or more spacer (e.g., targeting sequences, guide sequences, seed sequences, etc.). A “spacer” refers to a nucleic acid sequence that specifically binds (e.g., hybridizes) to or shares a region of complementarity with a target sequence. A spacer sequence may comprise between 5 and 50 nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, a spacer sequence comprises between 19 and 31 nucleotides. In some embodiments, a spacer sequence comprises 21, 22, or 23 nucleotides. In some embodiments, a multi-gRNA comprises one or more linking sequences (e.g., a linking polynucleotide). In some embodiments, the one or more linking sequences comprises one or more restriction endonuclease cleavage sites. In some embodiments, the cleavage sites are recognized by restriction endonucleases that create blunt-ended fragments. Examples of restriction endonucleases include BbsI, Bsal, Lgul, etc. Any of the promoter and / or regulatory sequences described herein can be incorporated into the isolated nucleic acid encoding one or more gRNAs. In some embodiments, the isolated nucleic acid for delivering the gRNA is an AAV vector. In some embodiments, the isolated nucleic acid for delivering the gRNA is a self-complementary AAV vector. In some embodiments, the gRNA can be delivered to the cell or subject using any suitable known method in the art, e.g., direct delivery by transfection, other viral platform such as lentivirus, adenovirus, HSV, ceDNA, retrovirus, liposome, nanoparticle, etc.).

[0094] Recombinant adeno-associated viruses (rAAVs)

[0095] Aspects of the disclosure relate to vectors comprising an isolated nucleic acid comprising a transgene encoding (i) a first region comprising a TadA peptide; (ii) a second region comprising a Cas protein (e.g., eNME2-C); and (iii) a third region comprising a gRNA targeting a mutant AAT protein. As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. In some embodiments, a vector is a viral vector, such as an rAAV vector, a lentiviral vector, an adenoviral vector, a retroviral vector, etc. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.

[0096] In some aspects, the disclosure provides isolated adeno-associated viruses (AAVs). As used herein with respect to AAVs, the term “isolated” refers to an AAV that has been artificially produced or obtained. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs.” Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s) (e.g., ocular tissues, neurons, liver, etc.). The AAV capsid is an important element in determining these tissue-specific targeting capabilities (e.g., tissue tropism). Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected.

[0097] Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003 / 0138772), the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner.

[0098] In some embodiments, an AAV capsid protein has a tropism for liver tissue (e.g., hepatocytes, etc.). In some embodiments, an AAV capsid protein does not target neuronal cells. In some embodiments, an AAV capsid protein does not cross the blood-brain barrier (BBB).

[0099] In some embodiments, an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.hr, AAVrh8, AAVrhlO, AAVrh39, AAVrh43, AAV.PHP.B, AAVPHP.eB, and variants of any of the foregoing. In some embodiments, an AAV capsid protein is of a serotype derived from a non-human primate, for example AAVrh8 serotype. In some embodiments, the AAV capsid protein is an AAV8 capsid protein.

[0100] In some embodiments, an rAAV vector or rAAV particle comprises a mutant ITR that lacks a functional terminal resolution site (TRS). The term “lacking a terminal resolution site” can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non- synonymous mutation, or mis sense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR). Without wishing to be bound by any particular theory, a rAAV vector comprising an ITR lacking a functional TRS produces a self- complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10): 1648-1656.

[0101] The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0102] In some embodiments, the disclosure relates to a host cell containing a nucleic acid comprising a transgene encoding (i) a first region comprising a TadA peptide; (ii) a second region comprising a Cas protein (e.g., eNME2-C); and (iii) a third region comprising a gRNA targeting a mutant AAT protein. A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. In some embodiments, the host cell is a hepatocyte.

[0103] The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

[0104] In some embodiments, recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with an AAV vector (comprising a transgene flanked by ITR elements) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the "AAV helper function" sequences (e.g., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein. The accessory function vector encodes nucleotide sequences for non- AAV derived viral and / or cellular functions upon which AAV is dependent for replication (e.g., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpes virus (other than herpes simplex virus type-1), and vaccinia virus. In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

[0105] As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

[0106] Methods

[0107] Methods for delivering a transgene (e.g., an isolated nucleic acid described herein) to a subject are provided by the disclosure. The methods typically involve administering to a subject an effective amount of an isolated nucleic acid encoding the transgene(s). In some embodiments, expression constructs described by the disclosure are useful for base editing in a cell or a subject. In some embodiments, expression constructs described by the disclosure are useful for treating diseases characterized by one or more G to A substitution in a protein of interest in a subject. In some embodiments, the disease is AAT deficiency.

[0108] In some embodiments, the method comprising administering to a subject in need thereof an effective amount of an isolated nucleic acid or an rAAV as described herein. A subject may be any mammalian organism, for example a human, non-human primate, horse, pig, dog, cat rodent, etc. In some embodiments a subject is a human.

[0109] An “effective amount” of a substance is an amount sufficient to produce a desired effect. In some embodiments, an effective amount of an isolated nucleic acid is an amount sufficient to transfect (or infect in the context of rAAV mediated delivery) a sufficient number of target cells of a target tissue of a subject. In some embodiments, a target tissue is CNS tissue (e.g., neurons, etc.) or liver cells (hepatocytes). In some embodiments, an effective amount of an isolated nucleic acid (e.g., which may be delivered via an rAAV) may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to correct the G to A substitution in a target protein, for example, the AAT gene, etc.), to extend the lifespan of a subject, to improve in the subject one or more symptoms of disease (e.g., a symptom of AAT deficiency), etc. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue as described elsewhere in the disclosure.

[0110] As used herein, the term “treating” refers to the application or administration of a composition encoding a transgene(s) to a subject, who has a genetic disorder caused by a G to A substitution in a responsible protein (e.g., AAT for AAT deficiency), with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, or the symptom of the disease. In some embodiments, the administration of a composition described herein reduces a level of a responsible protein (e.g., mutant AAT) by 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a reference value. Methods of measuring protein levels and / or activity are known in the art.

[0111] Alleviating a disease includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease (such as AAT deficiency) means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0112] "Development" or "progression" of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a disease includes initial onset and / or recurrence.

[0113] The isolated nucleic acids and rAAVs of the disclosure may be delivered to a subject in compositions according to any appropriate methods known in the art. For example, an rAAV, preferably suspended in a physiologically compatible carrier (z.e., in a composition), may be administered to a subject, i.e. host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some embodiments a host animal does not include a human. Delivery of the rAAVs to a mammalian subject may be by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue.

[0114] The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more guide RNAs). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.

[0115] Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

[0116] Optionally, the compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0117] The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intracerebroventricular, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.

[0118] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.

[0119] An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109to 1016genome copies. In some cases, a dosage between about 1011to 1013rAAV genome copies is appropriate. In certain embodiments, 1012or 1013rAAV genome copies is effective to target CNS tissue or liver tissue. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

[0120] In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than biweekly (e.g., once in a two-calendar week period). In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once per six calendar months. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

[0121] In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ~1013GC / ml or more). Methods for reducing aggregation of rAAVs are well known in the art and include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)

[0122] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

[0123] Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0124] In certain circumstances it will be desirable to deliver the rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver rAAVs. In some embodiments, a preferred mode of administration is by portal vein injection.

[0125] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0126] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0127] Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0128] The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0129] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0130] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0131] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

[0132] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

[0133] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.

[0134] Alternatively, nanocapsule formulations of the rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated.

[0135] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback- controlled delivery (U.S. Pat. No. 5,697,899). The invention also provides methods for expressing alpha 1-antitrypsin (AAT) protein in a subject. Typically, the subject has or is suspected of having an AAT deficiency. The methods typically involve administering to a subject an effective amount of a recombinant Adeno- Associated Virus (rAAV) harboring any of the isolated nucleic acids disclosed herein. In general, the “effective amount” of a rAAV refers to an amount sufficient to elicit the desired biological response. In some embodiments, the effective amount refers to the amount of rAAV effective for transducing a cell or tissue ex vivo. In other embodiments, the effective amount refers to the amount effective for direct administration of rAAV to a subject. As will be appreciated by those of ordinary skill in this art, the effective amount of the recombinant AAV of the invention varies depending on such factors as the desired biological endpoint, the pharmacokinetics of the expression products, the condition being treated, the mode of administration, and the subject. Typically, the rAAV is administered with a pharmaceutically acceptable carrier.

[0136] The subject may have a mutation in an AAT gene. The mutation may result in decreased expression of wild-type (normal) AAT protein. The subject may be homozygous for the mutation. The subject may be heterozygous for the mutation. The mutation may be a missense mutation. The mutation may be a nonsense mutation. The mutation may be a mutation listed in Table 1. The mutation may result in expression of a mutant AAT protein. The mutant protein may be a gain-of-function mutant or a loss-of-function mutant. The mutant AAT protein may be incapable of inhibiting protease activity. The mutant AAT protein may fail to fold properly. The mutant AAT protein may result in the formation of protein aggregates. The mutant AAT protein may result in the formation of intracellular AAT globules. The methods may also involve determining whether the subject has a mutation. Accordingly, the methods may involve obtaining a genotype of the AAT gene in the subject.

[0137] In some cases, after administration of the rAAV the level of expression of mutant AAT protein is determined in the subject. The administration may be performed on one or more occasions. When the administration is performed on one or more occasions, the level of mutant AAT in the subject are often determined after at least one administration. In some cases, the serum level of the mutant AAT in the subject is reduced by at least 85% following administration of the rAAV. The serum level of the mutant AAT in the subject may be reduced by at least 90% following administration of the rAAV. The serum level of the mutant AAT in the subject may be reduced by at least 95% following administration of the rAAV. However, in some cases, the serum level of the mutant AAT in the subject is reduced by at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% following administration of the rAAV. The reduction in the level of the mutant AAT may be sustained for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or more. In some cases, after 7 weeks of administration of the rAAV, the serum level of the mutant AAT is at a level of at least 50% compared with the serum level of the mutant AAT prior to administration of the rAAV. In certain cases, after 7 weeks of administration of the rAAV, the serum level of the mutant AAT is at a level of at least 75% compared with the serum level of the mutant AAT prior to administration of the rAAV.

[0138] In some instances, after administration of the rAAV at least one clinical outcome parameter associated with the AAT deficiency is evaluated in the subject. Typically, the clinical outcome parameter evaluated after administration of the rAAV is compared with the clinical outcome parameter determined at a time prior to administration of the rAAV to determine effectiveness of the rAAV. Often an improvement in the clinical outcome parameter after administration of the rAAV indicates effectiveness of the rAAV. Any appropriate clinical outcome parameter may be used. Typically, the clinical outcome parameter is indicative of the one or more symptoms of an AAT deficiency. For example, the clinical outcome parameter may be selected from the group consisting of: serum levels of the mutant AAT, presence of intracellular AAT globules, presence of inflammatory foci, breathing capacity, cough frequency, phlegm production, frequency of chest colds or pneumonia, and tolerance for exercise. Intracellular AAT globules or inflammatory foci are evaluated in tissues affected by the AAT deficiency, including, for example, lung tissue or liver tissue.

[0139] EXAMPLES

[0140] Example 1.

[0141] Different guide RNAs and ABE constructs were screened for (i) high on-target and low bystander editing; (ii) editing rate in PI*Z animals using AAV8; effect of editing on AATD associated liver disease; and effect of editing on AATD associated lung disease. AAT-null PI*Z mice were generated for the study. FIG. 1A is a figure showing the downstream effects caused by the Z allele (G-to-A mutation that causes a glutamic acid to lysine amino acid change at position 342 of AAT). FIG. IB is a figure showing the correction of the Z allele by an adenine base editor (reversion of the AAG codon back to GAG codon).

[0142] The HEK293T PI*Z reporter cell line was generated by transduction of HEK293T cells with lentivirus carrying a plasmid encoding for the Z-AAT coding sequence followed by puromycin selection. To test the editing efficiency of ABEs in editing the AAT site, a PI*Z reporter cell line was transfected with plasmids and quantified editing efficiency by targeted amplicon deep- sequencing. The transgene was packaged into AAV8 and injected into PI*Z mice via tail vein at 9 x 1011vg. Animals were sacrificed eight weeks after treatment. Five liver biopsies were taken per animal and the PI*Z site was sequenced using by targeted amplicon deep-sequencing. Two lobes were taken per animal to perform PAS-D staining. Sex-, age-, and weight- matched PI*Z mice were used as controls.

[0143] An all-in-one eNme2-C ABE8e / guide 2 plasmid achieved approximately 20% on target editing in HEK293T PI*Z cells, with bystander editing rates reaching as high as 12.83% depending on the adenine position (n=3). To minimize bystander editing, ABE9e was used, which has a narrower editing window than its predecessor, ABE8e. Indeed, eNme2-C ABE9e / guide 2 displayed lower bystander edits compared to ABE8e while maintaining high on-target editing. Its on-target editing rate averaged 21.08% while the highest bystander rate was 9.06% (n=3). An all- in-one eNme2-C ABE9e / guide 2 AAV was administered to PI*Z mice using AAV8 and showed approximately 30% editing efficiency after 8 weeks. PAS-D staining revealed a decrease in PAS- D+area and increase in PAS-D" cells in treated mice compared to control mice.

[0144] FIG. 2 provides a schematic of an exemplary AAV comprising a compact ABE utilizing an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2-C) and experiments following administration of said AAV to a mouse model subject (e.g., a disease model) to determine editing efficiency and disease progression.

[0145] FIG. 3A provides guide sequences and constructs shown with PI*Z mutation circled and PAM sequence underlined. FIG. 3B provides a summary of guides 1 and 2 editing efficiency in HEK293T PiZ reporter cells. Number represents mean editing rate measured with amplicon deep sequencing (n=3). FIG. 3C provides a graph showing the editing efficiency of an exemplary AAV comprising eNme2-C ABE8e. Data represents mean with SEM (n=3).

[0146] FIG. 4A provides a graph showing the editing efficiency of an exemplary AAV comprising eNme2-C ABE9e. Data represents mean with SEM (n=2). FIG. 4B provides a graph showing the ratio of bystander / on-target editing for ABE8e versus ABE9e. Analysis was done by dividing editing at each adenine position by A9 editing. Data represents mean with SEM (n=2 for ABE9e, n=3 for ABE8e).

[0147] FIG. 5A provides data from a deep sequencing data showing editing efficiency in mice treated for 4 or 8 weeks. Number represents mean editing rate (n=4 for 4- week timepoint, n=5 for 8-week timepoint). FIG. 5B provides a graph showing the allele percentage in PI*Z mice treated with ABE9e. Line represents median (n=4 for 4-week timepoint, n=5 for 8-week timepoint). FIG. 5C provides a representative image of PAS-D-stained liver section of untreated or treated mice. White circle shows PAS-D- area. Number of PAS-D- cells in a 20x field is also shown with the line representing median (n=3).

[0148] The inventors demonstrated the feasibility of an eNme2-C based ABE in single AAV to edit the PI*Z site. ABE9e showed high on-target edits with low bystander edits. Moreover, it was able to decrease PAS-D+globules in PI*Z mice. Future work would focus on assessing the therapeutic threshold needed to prevent lung emphysema development.

[0149] Example 2

[0150] Alpha- 1 antitrypsin deficiency (AATD) is commonly caused by a G-to-A mutation in the SERPINA1 gene (PiZ mutation). The mutant PiZ AAT protein is sequestered in hepatocytes, causing lung emphysema due to insufficient AAT protein to inhibit neutrophil elastase in the lung. This example describes that a compact adenine base editor (ABE) with evolved Cas9 nickase derived from Neisseria meningitidis (eNme2.C) can be packaged in a single AAV and correct the PiZ mutation in mouse models of AATD with high on-target and low-bystander edits. An all-in-one eNme2.C-TadA8e / guide 2 plasmid achieved approximately 20% on-target editing in PiZ reporter cells. TadA9e, which has a narrower editing window than TadA8e, reduced bystander editing without significantly affecting the on-target edit. In PiZ transgenic mice, eNme2.C-TadA9e AAV showed approximately 23% editing efficiency after 8 weeks and reduced liver disease burden in treated mice. In a new AAT-null;PiZ transgenic mouse model, ABE restored serum levels of AAT to beyond the 570 ug / mL therapeutic level. Moreover, ABE treatment was able to significantly correct lung functions in AAT-null;PiZ animals with emphysema. This example demonstrates the feasibility of an eNme2.C based ABE in single AAV to treat both AATD-associated liver and lung disease.

[0151] Molecular cloning

[0152] Plasmids expressing sgAAT were constructed using Gibson assembly using a gene block expressing the guide cassette and Addgene plasmid 122091. Plasmid expressing the guide RNA was cloned by Gibson assembly of gblock with the guide cassette and BfuAI digested Addgene 122091. Plasmid expressing the all-in-one eNme2.C-TadA8e was constructed by Gibson assembly using Addgene plasmid 121507 as a backbone, PCR-amplification of Addgene plasmid 185667 to retrieve eNme2.C-TadA8e, gene block containing Ula promoter, and PCR- amplification of sgAAT plasmid. Plasmid expressing the all-in-one eNme2.C-TadA9e was cloned by Gibson assembly of gblock with TadA9e with backbone from the all-in-one eNme2.C-TadA8e plasmid. Cell culture

[0153] Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (Gibco) and 1% (v / v) Penicillin / Streptomycin (Gibco). Cells were cultured at 37 °C with 5% CO2. HEK293T and 293fs cells were obtained from ATCC.

[0154] Generation of PiZ reporter line

[0155] The coding sequence of hAAT was packaged into lentivirus. Briefly, 293fs cells were plated into 6 wells to reach 70-80% confluency the next day. On the day of transfection, delta 8.2, VSV-G, and H633 were combined and transfected into cells using LT1.

[0156] For transduction, HEK293T cells were seeded into 6-well plates. The next day, virus, and polybrene were added. Puromycin was used to select transduced cells for 3 days. PiZ integration was verified through PCR amplification with primers and subsequent Sanger sequencing.

[0157] Transient transfection

[0158] For transfection, cells were plated on 12-well plates at 100,000 cells per well. The next day, cells were transfected with Lipofectamine 3000 (Invitrogen). For screening guides and Nme2 variants, 250ng of the effector and 83ng of the sgRNA were transfected. For testing all- in-one constructs, lOOOng of the plasmid was transfected into the PiZ reporter cell line. 3 days after transfection, genomic DNA was collected with quick extraction buffer (Epicenter). The lysate was incubated at 65 °C for 15 minutes, and 98 °C for 5 minutes.

[0159] Off-target analysis

[0160] Off-target analysis was performed with Cas-OFFinder using 5 or fewer mismatches relative to the intended target site. To determine potential off-target sites, sites with appropriate PAMs were filtered for, followed by sites with conserved seed regions. Sites of known genes were then sequenced with amplicon sequencing.

[0161] Amplicon sequencing and. data analysis

[0162] Sequencing library preparation was done by standard techniques. For the first round of PCR, primers with Illumina forward and reverse adapters were used to amplify the gene of interest using either lul cell culture lysate or lOOng mouse DNA. PCR was performed with Phusion Flash High-Fidelity PCR Master Mix at 98 °C for 10 s, then 20 cycles of 98 °C for 1 s, 55 °C for 5 s, and 72 °C for 7s, followed by a final 72 °C extension for 1 min. lul of PCR product was then used for the second round of PCR in which a unique Illumina barcode was added to each sample. As before, PCR was performed at 98 °C for 1 s, 55 °C for 5 s, and 72 °C for 7s, followed by a final 72 °C extension for 1 min. PCR 2 products were purified by gel extraction using the QIAquick Gel Extraction Kit (Qiagen) and quantified by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The library was sequenced on an Illumina MiniSeq instrument following the manufacturer’s protocols. Sequencing reads were demultiplexed using bcl2fastq (Illumina). To quantify the frequency of precise editing and indels, CRISPResso2 was run in base editor mode with ‘min_average_read_quality’ > 30.

[0163] The ratio of on-target to bystander edit was computed by: editing percentage at bystander adenine / editing percentage at target adenine.

[0164] AAV production

[0165] AAV were produced using standard techniques.

[0166] Mouse experiments

[0167] AAV was administered via tail- vein injection for treated animals. AAT-null / PiZ animals were generated by crossing the AAT-null and PiZ animals. The human AAT gene was verified via genomic DNA PCR (5’ TTG AGG AGC GAG AGG CAG TT (SEQ ID NO: 10), 5’ GAG GCG CTT GTC AGG AAG AT (SEQ ID NO: 11)).

[0168] For the PiZ animal study, both male and female 4-8-week-old animals were used. For the AAT-null;PiZ animal study, both male and female animals were used for the PBS, LPS and ABE + LPS group (9-16 week-old, 13-18 week-old, and 5-10-week-old animals, respectively). No animals were excluded from the study. PiZ animals were taken down at either 4 or 8 weeks post AAV treatment while AAT-null;PiZ animals were taken down 15 weeks post AAV treatment. 5 liver biopsies were harvested per animal and genomic DNA was collected using DNeasy Blood & Tissue Kit (Qiagen, 69506). Editing efficiency was computed by taking the average across all five lobes. For histology, liver lobes were fixed in 10% neutral buffered formalin overnight before being embedded in paraffin. Fixed sections were stained with PAS-D to visualize AAT globules. PAS-D staining was analyzed using QuPath color thresholding.

[0169] ELISA

[0170] Animals were bled via facial vein to retrieve serum, and ELISA was performed as previously described. To detect hAAT, clear flat-bottom immuno nonsterile 96-Well Plates (Thermo Fisher Scientific, 3855) were coated with goat anti-human AAT antibody (Bethyl Laboratories, A80-122A) overnight. The next morning, plates were blocked with Intercept PBS blocking buffer (Licorbio, 927-70003) followed by the addition of serially diluted samples and standard curves (Athens research, 16-16-011609) in triplicates. The plate was incubated for an hour, and goat anti-human AAT antibody (Bethyl Laboratories, A80-122P) was added for one hour. Between each step, the plate was washed with PBST. 3,3’,5,5’-tetramethylbenzidine (TMB) peroxidase (Thermo Fisher Scientific, 50-674-21) was added to the wells and the reaction was stopped by adding 0.18M sulfuric acid. The plate was read at wavelength 450nm.

[0171] LPS treatment

[0172] Animals were either treated with AAV or untreated at week 0, followed by lipopolysaccharides (LPS) or PBS challenges at weeks 8, 10, and 12. For LPS challenges, mice were anesthetized with an i.p. dose of ketamine / xylazine mixture (90 mg / kg and 4.5 mg / kg, respectively), then intratracheally cannulated with a 22-gauge angiocatheter using a mouse intubating board. LPS isolated from Escherichia coli O55:B5 (Cayman Chemical, item number 19660, batch 0710200-1) were administered (30 pL, 1 pg / mL in PBS) via direct injection through the angiocatheter during inspiration. Following instillation, the animals received 2-3 ventilations of air to ensure the solution was properly inhaled. After the LPS challenge, the mice were allowed to recover and return to the colony.

[0173] FlexiVent

[0174] First, animals were anesthetized with a ketamine / xylazine mixture (100 mg / kg and 10 mg / kg, i.p.). A tracheotomy was performed to insert an 18-gauge metal cannula, and the animal was connected to the machine. Pulmonary function was assessed through the Forced oscillation technique (FOT) performed using the flexiVent FX system (SCIREQ Inc., Montreal Qc, Canada). Mice were ventilated with a tidal volume of 10 mL / kg, a frequency of 150 breaths / min, an inspiratory to expiratory ratio of 2:3, and a positive end-expiratory pressure of 3 cmH20. Then, two deep inflations were given to maximally inflate the lungs and standardize lung volume. Inspiratory capacity (IC), compliance (C), elastance (Ers), and resistance (Rrs) were calculated from volume, pressure and flow signals collected during the test. The flow signal is derived from the volume signal. Pressure- volume curve was generated using a ramp-style pressure-driven maneuver (PVr-P). Static compliance (Cst) was measured using the PV loop between the pressures of 3 -7 cmlLO. Each measurement was taken three times, and the average was used for further analysis.

[0175] Data

[0176] To edit the PiZ site, a compact ABE system that can be packaged into a single adeno- associated virus (AAV) was developed. The evolved Neisseria meningitidis Cas9 (eNme2.C), which has been shown to have high base editing capabilities, was used. Moreover, it is ~3.2kb, thus small enough to be packaged into a single AAV for therapeutic delivery. First, two guides compatible with an eNme2.C Cas9 ABE system, which uses the N4C PAM, were tested. The two sgRNAs (referred to as guide 1 or 2) have slightly offset editing (FIGs. 6A and 6B; Table 1).

[0177] Table 1

[0178] To study PiZ allele editing events by ABEs, HEK293T cells were transduced with a lentiviral construct with the coding sequence of the Z allele, generating a PiZ reporter line. The PiZ reporter cells were transduced with constructs encoding eNme2.C-TadA8e and one of two sgRNAs (guide 1 or 2) and high-throughput sequencing was performed using primers specific to the reporter that cannot amplify the endogenous SERPINA1 gene. For guide 2, the target A in PiZ is at position 9 (A9). To directly compare the editing rate, the same “A” positions for guides 1 and 2 were defined. Guides 1 and 2 showed -36.98% and -33.84% editing, respectively (FIG. 6C). Although guide 1 showed slightly higher on-target editing at the A9 site, the bystander rates were equally high - bystander editing ranged from -0.03% to -33.26% compared to the -0.03% to -12.19% with guide 2. Given the lower bystander rate but comparable on-target editing rate of guide 2, guide 2 was chosen for subsequent experiments.

[0179] Both eNme2.C and guide 2 were into a single vector (referred to herein as “eNme2.C- TadA8e”) driven by the ula promoter. eNme2.C-TadA8e showed an average of -23.18% on- target editing at the A9 position, with bystander rates ranging from -4.16% to 14.13% (FIG. 6E).

[0180] Although some bystander edits have been characterized as not affecting AAT protein function or serum secretion, it was one objective to reduce these bystander edits further, as they can generate mutant AAT variants with unknown function and cause an unknown immune response. TadA9e is a derivative of TadA8e but with a narrower editing window due to weaker TadA-DNA interaction. To test its activity, the TadA8e domain was swapped with TadA9e, thereby generating the “all-in-one eNme2.C-TadA9e”. This all-in-one vector proved to have efficient in vitro editing, with on-target editing averaging -17.94% (FIG. 7A). When bystander editing was normalized to on-target A9 editing, eNme2.C-TadA9e showed lower bystander editing compared to eNme2.C-TadA8e and had more perfect alleles (FIGs. 7B and 7C). Thus, the all-in-one eNme2.C-TadA9e was evaluated in subsequent experiments.

[0181] Nme2-based editors have higher fidelity compared to the more widely used SpyCas9. Despite this, it was an objective to characterize the off-target editing of eNme2.C-TadA9e through computational and experimental approaches. First, the endogenous wild-type PiM site in the PiZ reporter cell transfected with the all-in-one eNme2.C-TadA8e was sequenced to detect any editing. The absence of any editing at the PiM site, which differs from the PiZ site by a single nucleotide, points to the high fidelity of the system described in this example (FIG. 6F and 10).

[0182] Cas9-based editing modalities can induce both Cas-independent and -dependent off- target edits. Cas-dependent off-target editing is due to sequence similarities between the target protospacer and off-target protospacer, allowing the guide to bind and hence editing to occur. Cas-independent off-targeting editing is due to long-term expression of the TadA deaminase enzyme, causing low-level deamination across the genome and RNA. The Cas-dependent off- targets were characterized with Cas-OFFinder. The Cas-OFFinder nominated 150 sites with five or fewer mismatches to the target protospacer. After filtering for suitable PAM sites, and sites with conserved seed regions, defined as nucleotides 17-23, the editing rate was tested at five sites by high-throughput sequencing (Table 2). There was no difference in editing between the control and transfected cells (FIG. 7D).

[0183] Table 2

[0184] The all-in-one eNme2.C-TadA9e was then tested in PiZ animals, which carry the human

[0185] PiZ transgene integrated. The vector was packaged into AAV8, and 9el Ivg was delivered to animals via tail- vein injection. Control animals were left untreated. After 4 or 8 weeks, the liver of each animal was collected, and the PiZ site was amplified for targeted amplicon sequencing (FIG. 8A). At 4 weeks, on-target editing was -16.75% and this increased to 22.83% at 8 weeks (FIG. 8B). Allele analysis showed that in the treated animals, perfect editing (K342E allele, A9G) represented 12.6% of the allele population at 4 weeks, and -18.6% at 8 weeks (FIG. 8C). The D341G + K342E (A7G+A9G) allele and D341G (A7G) allele were the other top alleles, representing 1.62% and 2.08%, respectively, of the allele population at 8 weeks (FIG. 8C).

[0186] These data show that the perfectly edited allele presents the majority of the edited alleles. To test if eNme2.C-TadA8e also has high activity in vivo, eNme2.C-TadA8e was packaged into AAV8 and injected 8el Ivg into PiZ animals. Eight weeks after treatment, mouse liver DNA was collected, and the PiZ site was sequenced. Editing at the target A9 was -15.66% (FIG. 11). The high on-target editing rate regardless of the deaminase indicates that the Cas9 enzyme is likely very active in vivo.

[0187] It was previously reported that significant base editing correction of the PiZ site can lead to decreased AAT globules in the liver, which can be detected with Periodic acid-Schiff- diastase stain (PAS-D stain). It was investigated whether the editing rate of constructs described herein was sufficient and performed PAS-D staining on mouse liver. Indeed, animals treated for 8 weeks showed a significant decrease in PAS-D positive area (p=0.04) (FIG. 8D).

[0188] The PiZ animal carries multiple copies of the human Z-AAT allele, along with five isoforms of the murine Serpinal gene. Due to the presence of its endogenous Serpinal genes, the PiZ animal does not develop emphysema. The five copies of the Serpinal gene were knocked out in C57BL / 6J animals to generate AAT-null mice, which develop emphysema both spontaneously and through LPS challenges. To study the therapeutic advantage of ABE for correcting human Z-AAT, the AAT-null animals were crossed with PiZ animals to generate AAT-null;PiZ (FIG. 9A). Given that this strain carries multiple copies of the human Z-AAT allele, but no copies of murine Serpinal, it will develop both liver and lung disease.

[0189] The editing efficiency of this system was tested in the AAT-null;PiZ animal. Animals were injected with 9el lvg AAV and followed for 15 weeks. Treated animals showed -30.78% on-target editing at 15 weeks (FIG. 9A) with perfect allele reaching 23.10% (FIG. 13). Serum was collected from animals at 8, 12, and 15 weeks to test for human AAT. It is estimated that serum AAT level below 1 luM (570 ug / mL) significantly increases the risk of emphysema development. Because there was no suitable M-AAT specific antibody for ELISA, an antibody recognizing both Z- and M-AAT was used. In treated animals, total AAT levels increased to an average of -1,321.7 ug / mL at 8 weeks and consistently stayed above 570 ug / mL throughout the 15-week timeline (FIG. 9B, Table 3). To estimate the level of edited AAT, the majority of which likely is the M-AAT wild-type variant, the AAT levels were subtracted from the untreated animals, which only express the Z-AAT protein. At 15 weeks, the differential AAT level in treated animals subtracted from control was 909 ug / mL on average (FIG. 12).

[0190] Table 3

[0191] AAT-null mice have been shown to manifest lung disease with pulmonary physiology consistent with an emphysema phenotype, but only after prolonged aging or exposure to a proinflammatory stimulus or tobacco smoke. Proinflammatory stimuli are thought to accelerate the development of emphysema in the AAT deficient state by recruiting neutrophils to the lung, triggering their release of NE in proximity to pulmonary interstitial elastin fibers. Without sufficient AAT to counteract NE, destruction of the extracellular matrix of the lung leads to hyperinflation (increased inspiratory capacity) and decreased pulmonary elastic recoil, with a resultant decrease in elastance and lung tissue resistance, and an increase in lung compliance - a picture that matches experimentally induced emphysema in mice. To demonstrate the therapeutic effect of base editing on the lung disease phenotype, pulmonary physiology was assessed with the FlexiVent system in AAT-null;PiZ animals with or without LPS challenge, and in the base-edited mice with LPS challenge. The LPS challenge induced the expected emphysema physiology, consisting of increased inspiratory capacity (IC), increased static compliance (Cst), decreased elastance of the respiratory system (Ers) and decreased respiratory system resistance (Rrs) (FIGs. 9C-G). Each of these parameters was corrected by base editing to a statistically significant extent (FIGs. 9C-9G).

[0192] Alpha- 1 antitrypsin deficiency affects approximately 100,000 people in the United States every year. Although AAT-specific therapies, such as AAT protein infusion have been clinically successful, they still have specific drawbacks such as the need for weekly, costly treatments. Moreover, the only FDA-approved treatment for AATD-associated liver disease is organ transplant. A one-time therapy targeting the genetic cause can address these drawbacks.

[0193] As disclosed herein, the therapeutic potential of a novel editing agent (e.g., eNme2.C- TadA9e) was evaluated for treating AATD caused by the PiZ allele. The ABE herein was systematically optimized for minimal bystander and maximal on-target editing and assessed the off-target editing rate of our system through both computational and experimental validation. The lack of editing at the endogenous PiM site in the PiZ reporter cell suggests that ABE would not cause bystander editing in patients with the Pi*Z genotype. The system provided to be highly efficient in editing the PiZ allele across two animal models carrying the human allele, with editing reaching 30.78% by week 15. Notably, the AAV dosage, 9el lvg or 4.5el3vg / kg, is significantly lower than typical doses in clinical trials. Given that high AAV doses can cause toxicity, the low dosage further underscores eNme2.C-TadA9e as a promising candidate for AATD therapy. The editing percentage could continue to improve, as cells with the PiM allele have a survival advantage over cells with the PiZ allele, and the ABE system may continue to edit the PiZ site. This is supported by the observation that editing steadily increased from 16.75% to 30.78% by week 15. Importantly, treated animals have a significant reduction in AAT globule accumulation - a key factor in the development of AATD-associated liver fibrosis.

[0194] To study the effect of editing on AATD-associated lung emphysema, a new AATD mouse model was generated by crossing the PiZ and AAT-null strain. The AAT-null;PiZ strain was able to recapitulate emphysema through sequential LPS doses. AAT-null;PiZ animals treated with ABE for 8 weeks had a total serum AAT level of -1,322 pg / mL, surpassing the 570 pg / mL threshold needed for decreased risk of lung emphysema development. Finally, the therapeutic potential of ABE for resolving AATD-associated emphysema was assessed. After establishing a model of emphysema with the AAT-null;PiZ animals, the animals were treated with ABE. ABE+LPS treated AAT-null;PiZ animals demonstrated a significant reduction in inspiratory capacity, compliance, and an increase in elastance and resistance.

[0195] REPRESENTATIVE SEQUENCES

[0196] > TadA-8e peptide amino acid sequence (SEQ ID NO: 1) MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAH AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAA GSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0197] > TadA-9e peptide amino acid sequence (SEQ ID NO: 2) MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAH

[0198] AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKTGAAG

[0199] SLMNVLNYPGMKHRVEITEGILADECAALLCDFYRMPRRVFNAQKKAQSSIN

[0200] > Neisseria meningitidis evolved Cas9 nickase (eNme2.C) amino acid sequence (SEQ ID NO:

[0201] 3)

[0202] MAAFKSNPINYILGLAIGIASVGWAMVEIDEEGNPIRLIDLGVRVFERAEVPKTGDSLAM

[0203] ARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLITSLPNTPWQLRAAAL

[0204] DRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETAAKELGALLKGVANNAHALQTGDFR

[0205] TPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGI

[0206] ETLLMTQRPALSGDAVQKMLGHCTLEPTEPKAAKNTYTAERFIWLTKLNNLRILEQGSE

[0207] RPLTDTERSTLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMK

[0208] AYHAISRALEKEGLKDKKSPLNLSSELQDEIGTAFSLFKTDEDITGRLKDRVQPEILEALL

[0209] KHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGVHYGKKNTEEKIYLPPIPADEIR

[0210] NPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIAKRQEENRKDREK

[0211] AAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALP

[0212] FSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPSSKK

[0213] QRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVVASNGQITNLLRG

[0214] FWRLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTVDKETGK

[0215] VLYQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHE

[0216] YVTPLFVSRAPNRKMSGAHKDTLRSAKRFVKHNEKISVKRVWLTEIKLADLENMVNY

[0217] KNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAVRVEKTQKSGVLL

[0218] NKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDD

[0219] SYTFCFSLHKYDLIAFQKDEKSKVEFAYYINCDSSSGGFYLAWHDKGSREQRFRISTQNL

[0220] ALIQKYQVNELGKEIRPCRLKKRPPVR

[0221] > AAT gRNA 1 nucleic acid sequence (SEQ ID NO: 4) tgaccatcgacaagaaagggact

[0222] > AAT gRNA 2 nucleic acid sequence (SEQ ID NO: 5) ccatcgacAagaaagggactgaa

[0223] > AAT gRNA 3 nucleic acid sequence (SEQ ID NO: 6) Atcgacaagaaagggactga

[0224] > Mutant alpha 1 antitrypsin (AAT) nucleic acid sequence (SEQ ID NO: 7) tgggcaggaactgggcactgtgcccagggcatgcactgcctccacgcagcaaccctcagagtcctgagctgaaccaagaaggaggag ggggtcgggcctccgaggaaggcctagccgctgctgctgccaggaattccaggttggaggggcggcaacctcctgccagccttcaggc cactctcctgtgcctgccagaagagacagagcttgaggagagcttgaggagagcaggaaaggtgggacattgctgctgctgctcactcag ttccacaggtgggagggacagcagggcttagagtgggggtcattgtgcagatgggaaaacaaaggcccagagaggggaagaaatgcc caggagctaccgagggcaggcgacctcaaccacagcccagtgctggagctgtgagtggatgtagagcagcggaatatccattcagcca gctcaggggaaggacaggggccctgaagccaggggatggagctgcagggaagggagctcagagagaaggggaggggagtctgag ctcagtttcccgctgcctgaaaggagggtggtacctactcccttcacagggtaactgaatgagagactgcctggaggaaagctcttcaagtg tggcccaccccaccccagtgacaccagcccctgacacgggggagggagggcagcatcaggaggggctttctgggcacacccagtacc cgtctctgagctttccttgaactgttgcattttaatcctcacagcagctcaacaaggtacataccgtcaccatccccattttacagatagggaaat tgaggctcggagcggttaaacaactcacctgaggcctcacagccagtaagtgggttccctggtctgaatgtgtgtgctggaggatcctgtg ggtcactcgcctggtagagccccaaggtggaggcataaatgggactggtgaatgacagaaggggcaaaaatgcactcatccattcactct gcaagtatctacggcacgtacgccagctcccaagcaggtttgcgggttgcacagcgggcgatgcaatctgatttaggcttttaaagggattg caatcaagtggggccccactagcctcaaccctgtacctcccctcccctccacccccagcagtctccaaaggcctccaacaaccccagagt gggggccatgtatccaaagaaactccaagctgtatacggatcacactggttttccaggagcaaaaacagaaacaggcctgaggctggtca aaattgaacctcctcctgctctgagcagcctggggggcagactaagcagagggctgtgcagacccacataaagagcctactgtgtgccag gcacttcacccgaggcacttcacaagcatgcttgggaatgaaacttccaactctttgggatgcaggtgaaacagttcctggttcagagaggt gaagcggcctgcctgaggcagcacagctcttctttacagatgtgcttccccacctctaccctgtctcacggccccccatgccagcctgacg gttgtgtctgcctcagtcatgctccatttttccatcgggaccatcaagagggtgtttgtgtctaaggctgactgggtaactttggatgagcggtc tctccgctctgagcctgtttcctcatctgtcaaatgggctctaacccactctgatctcccagggcggcagtaagtcttcagcatcaggcattttg gggtgactcagtaaatggtagatcttgctaccagtggaacagccactaaggattctgcagtgagagcagagggccagctaagtggtactct cccagagactgtctgactcacgccaccccctccaccttggacacaggacgctgtggtttctgagccaggtacaatgactcctttcggtaagt gcagtggaagctgtacactgcccaggcaaagcgtccgggcagcgtaggcgggcgactcagatcccagccagtggacttagcccctgttt gctcctccgataactggggtgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacggacgaggac agggccctgtctcctcagcttcaggcaccaccactgacctgggacagtgaatcgtaagtatgcctttcactgcgagaggttctggagaggct tctgagctccccatggcccaggcaggcagcaggtctggggcaggaggggggttgtggagtgggtatccgcctgctgaggtgcagggca gatggagaggctgcagctgagctcctattttcataataacagcagccatgagggttgtgtcctgtttcccagtcctgcccggtcccccctcgg tacctcctggtggatacactggttcctgtaagcagaagtggatgagggtgtctaggtctgcagtcctggcaccccaggatgggggacacca gccaagatacagcaacagcaacaaagcgcagccatttctttctgtttgcacagctcctctgtctgtcgggggctcctgtctgttgtctcctataa gcctcaccacctctcctactgcttgggcatgcatctttctccccttctatagatgaggaggttaaggtccagagaggggtggggaggaacgc cggctcacattctccatcccctccagatatgaccaggaacagacctgtgccaggcctcagccttacatcaaaatgggcctccccatgcacc gtggacctctgggccctcctgtcccagtggaggacaggaagctgtgaggggcactgtcacccagggctcaagctggcattcctgaataat cgctctgcaccaggccacggctaagctcagtgcgtgattaagcctcataaccctccaaggcagttactagtgtgattcccattttacagatga ggaagatggggacagagaggtgaataactggccccaaatcacacaccatccataattcgggctcaggcacctggctccagtccccaaac tcttgaacctggccctagtgtcactgtttctcttgggtctcaggcgctggatggggaacaggaaacctgggctggacttgaggcctctctgat gctcggtgacttcagacagttgctcaacctctctgttctcttgggcaaaacatgataacctttgacttctgtcccctcccctcaccccacccgac cttgatctctgaagtgttggaaggatttaatttttcctgcactgagttttggagacaggtcaaaaagatgaccaaggccaaggtggccagtttc ctatagaacgcctctaaaagacctgcagcaatagcagcaagaactggtattctcgagaacttgctgcgcagcaggcacttcttggcattttat gtgtatttaatttcacaatagctctatgacaaagtccacctttctcatctccaggaaactgaggttcagagaggttaagtaacttgtccaaggtca cacagctaatagcaagttgacgtggagcaatctggcctcagagcctttaattttagccacagactgatgctcccctcttcatttagccaggctg cctctgaagttttctgattcaagacttctggcttcagctttgtacacagagatgattcaatgtcaggttttggagtgaaatctgtttaatcccagac aaaacatttaggattacatctcagttttgtaagcaagtagctctgtgatttttagtgagttatttaatgctctttggggctcaatttttctatctataaaa tagggctaataatttgcaccttatagggtaagctttgaggacagattagatgatacggtgcctgtaaaacaccaggtgttagtaagtgtggca atgatggtgacgctgaggctgatgtttgcttagcatagggttaggcagctggcaggcagtaaacagttggataatttaatggaaaatttgcca aactcagatgctgttcactgctgagcaggagccccttcctgctgaaatggtcctggggagtgcagcaggctctccgggaagaaatctacca tctctcgggcaggagctcaacctgtgtgcaggtacagggagggcttcctcacctggtgcccactcatgcattacgtcagttattcctcatccc tgtccaaaggattcttttctccattgtacagctatgaagctagtgctcaaagaagtgaagtcatttaccccaggccccctgccagtaagtgaca gggcctggtcacacttgggtttatttattgcccagttcaacaggttgtttgaccataggcgagattctcttccctgcaccctgccgggttgctctt ggtcccttattttatgctcccgggtagaaatggtgtgagattaggcagggagtggctcgcttccctgtccctggccccgcaaagagtgctcc cacctgccccgatcccagaaatgtcaccatgaagccttcattcttttggtttaaagcttggcctcagtgtccgtacaccatggggtacttggcc agatggcgactttctcctctccagtcgccctcccaggcactagcttttaggagtgcagggtgctgcctctgatagaagggccaggagagag caggttttggagtcctgatgttataaggaacagcttgggaggcataatgaacccaacatgatgcttgagaccaatgtcacagcccaattctga cattcatcatctgagatctgaggacacagctgtctcagttcatgatctgagtgctgggaaagccaagacttgttccagctttgtcactgacttgc tgtatagcctcaacaaggccctgaccctctctgggcttcaaactcttcactgtgaaaggaggaaaccagagtaggtgatgtgacaccagga aagatggatgggtgtgggggaatgtgctcctcccagctgtcaccccctcgccaccctccctgcaccagcctctccacctcctttgagccca gaattcccctgtctaggagggcacctgtctcatgcctagccatgggaattctccatctgttttgctacattgaacccagatgccattctaaccaa gaatcctggctgggtgcaggggctctcgcctgtaaccccagcactttgggaggccaaggcaggcggatcaagaggtcaggagttcaaga cctgcctggccaacacggtgaaacctcagctctactaaaaatacaaaaattagccaggcgtggtggcacacgcctgtaatcccagctatttg ggaagctgagacagaagaatttcttgaacccgggaggtggaggtttcagtgagccgagatcacgccactgcactccaccctggcagataa agcgagactctgtctcaaaaaaaacccaaaaacctatgttagtgtacagagggccccagtgaagtcttctcccagccccactttgcacaact ggggagagtgaggccccaggaccagaggattcttgctaaaggccaagtggatagtgatggccctgccagggctagaagccacaacctc tggccctgaggccactcagcatatttagtgtccccaccctgcagaggcccaactccctcctgaccactgagccctgtaatgatgggggaatt tccataagccatgaaggactgcacaaagttcagttgggaagtgaaagagaaattaaagggagatggaaatatacagcactaattttagcac cgtctttagttctaacaacactagctagctgaagaaaaatacaaacatgtattatgtaatgtgtggtctgttccatttggattacttagaggcacg agggccaggagaaaggtggtggagagaaaccagctttgcacttcatttgttgctttattggaaggaaacttttaaaagtccaagggggttga agaatctcaatatttgttatttccagctttttttctccagtttttcatttcccaaattcaaggacacctttttctttgtattttgttaagatgatggttttggtt ttgtgactagtagttaacaatgtggctgccgggcatattctcctcagctaggacctcagttttcccatctgtgaagacggcaggttctacctag ggggctgcaggctggtggtccgaagcctgggcatatctggagtagaaggatcactgtggggcagggcaggttctgtgttgctgtggatga cgttgactttgaccattgctcggcagagcctgctctcgctggttcagccacaggccccaccactccctattgtctcagccccgggtatgaaac atgtattcctcactggcctatcacctgaagcctttgaatttgcaacacctgccaacccctccctcaaaagagttgccctctcagatccttttgatg taaggtttggtgttgagacttatttcactaaattctcatacataaacatcactttatgtatgaggcaaaatgaggaccagggagatgaatgacttg tcctggctcatacacctggaaagtgacagagtcagattagatcccaggtctatctgaagttaaaagaggtgtcttttcacttcccacctcctcc atctactttaaagcagcacaaacccctgctttcaaggagagatgagcgtctctaaagcccctgacagcaagagcccagaactgggacacc attagtgacccagacggcaggtaagctgactgcaggagcatcagcctattcttgtgtctgggaccacagagcattgtggggacagccccgt ctcttgggaaaaaaaccctaagggctgaggatccttgtgagtgttgggtgggaacagctcccaggaggtttaatcacagcccctccatgctc tctagctgttgccattgtgcaagatgcatttcccttctgtgcagcagtttccctggccactaaatagtgggattagatagaagccctccaaggg cttccagcttgacatgattcttgattctgatctggcccgattcctggataatcgtgggcaggcccattcctcttcttgtgcctcattttcttcttttgta aaacaatggctgtaccatttgcatcttagggtcattgcagatgtaagtgttgctgtccagagcctgggtgcaggacctagatgtaggattctgg ttctgctacttcctcagtgacattgaatagctgacctaatctctctggctttggtttcttcatctgtaaaagaaggatattagcattagcacctcacg ggattgttacaagaaagcaatgaattaacacatgtgagcacggagaacagtgcttggcatatggtaagcactacgtacattttgctattcttct gattctttcagtgttactgatgtcggcaagtacttggcacaggctggtttaataatccctaggcacttccacgtggtgtcaatccctgatcactg ggagtcatcatgtgccttgactcggggcctggcccccccatctctgtcttgcaggacaatgccgtcttctgtctcgtggggcatcctcctgct ggcaggcctgtgctgcctggtccctgtctccctggctgaggatccccagggagatgctgcccagaagacagatacatcccaccatgatca ggatcacccaaccttcaacaagatcacccccaacctggctgagttcgccttcagcctataccgccagctggcacaccagtccaacagcac caatatcttcttctccccagtgagcatcgctacagcctttgcaatgctctccctggggaccaaggctgacactcacgatgaaatcctggaggg cctgaatttcaacctcacggagattccggaggctcagatccatgaaggcttccaggaactcctccgtaccctcaaccagccagacagccag ctccagctgaccaccggcaatggcctgttcctcagcgagggcctgaagctagtggataagtttttggaggatgttaaaaagttgtaccactc agaagccttcactgtcaacttcggggacaccgaagaggccaagaaacagatcaacgattacgtggagaagggtactcaagggaaaattg tggatttggtcaaggagcttgacagagacacagtttttgctctggtgaattacatcttctttaaaggtaaggttgctcaaccagcctgagctgttc ccatagaaacaagcaaaaatattctcaaaccatcagttcttgaactctccttggcaatgcattatgggccatagcaatgcttttcagcgtggatt cttcagttttctacacacaaacactaaaatgttttccatcattgagtaatttgaggaaataatagattaaactgtcaaaactactgacagctctgca gaacttttcagagcctttaatgtccttgtgtatactgtatatgtagaatatataatgcttagaactatagaacaaattgtaatacactgcataaagg gatagtttcatggaacatactttacacgactctagtgtcccagaatcagtatcagttttgcaatctgaaagacctgggttcaaatcctgcctctaa cacaattagcttttgacaaaaacaatgcattctacctctttgaggtgctaatttctcatcttagcatggacaaaataccattcttgctgtcaggttttt ttaggattaaacaaatgacaaagactgtggggatggtgtgtggcatacagcaggtgatggactcttctgtatctcaggctgccttcctgcccc tgaggggttaaaatgccagggtcctgggggccccagggcattctaagccagctcccactgtcccaggaaaacagcataggggagggga ggtgggaggcaaggccaggggctgcttcctccactctgaggctcccttgctcttgaggcaaaggagggcagtggagagcagccaggct gcagtcagcacagctaaagtcctggctctgctgtggccttagtgggggcccaggtccctctccagccccagtctcctccttctgtccaatga gaaagctgggatcaggggtccctgaggcccctgtccactctgcatgcctcgatggtgaagctctgttggtatggcagaggggaggctgct caggcatctgcatttcccctgccaatctagaggatgaggaaagctctcaggaatagtaagcagaatgtttgccctggatgaataactgagct gccaattaacaaggggcagggagccttagacagaaggtaccaaatatgcctgatgctccaacattttatttgtaatatccaagacaccctcaa ataaacatatgattccaataaaaatgcacagccacgatggcatctcttagcctgacatcgccacgatgtagaaattctgcatcttcctctagtttt gaattatccccacacaatctttttcggcagcttggatggtcagtttcagcaccttttacagatgatgaagctgagcctcgagggatgtgtgtcgt caagggggctcagggcttctcagggaggggactcatggtttctttattctgctacactcttccaaaccttcactcacccctggtgatgcccacc ttcccctctctccaggcaaatgggagagaccctttgaagtcaaggacaccgaggaagaggacttccacgtggaccaggtgaccaccgtg aaggtgcctatgatgaagcgtttaggcatgtttaacatccagcactgtaagaagctgtccagctgggtgctgctgatgaaatacctgggcaat gccaccgccatcttcttcctgcctgatgaggggaaactacagcacctggaaaatgaactcacccacgatatcatcaccaagttcctggaaaa tgaagacagaaggtgattccccaacctgagggtgaccaagaagctgcccacacctcttagccatgttgggactgaggcccatcaggactg gccagagggctgaggagggtgaaccccacatccctgggtcactgctactctgtataaacttggcttccagaatgaggccaccactgagttc aggcagcgccatccatgctccatgaggaggacagtacccaggggtgaggaggtaaaggtctcgtccctggggacttcccactccagtgt ggacactgtcccttcccaatatccagtgcccagggcagggacagcagcaccaccacacgttctggcagaaccaaaaaggaacagatgg gcttcctggcaaaggcagcagtggagtgtggagttcaagggtagaatgtccctggggggacgggggaagagcctgtgtggcaaggccc agaaaagcaaggttcggaattggaacagccaggccatgttcgcagaaggcttgcgtttctctgtcactttatcggtgctgttagattgggtgtc ctgtagtaagtgatacttaaacatgagccacacattagtgtatgtgtgtgcattcgtgattatgcccatgccctgctgatctagttcgttttgtaca ctgtaaaaccaagatgaaaatacaaaaggtgtcgggttcataataggaatcgaggctggaatttctctgttccatgccagcacctcctgaggt ctctgctccaggggttgagaaagaacaaagaggctgagagggtaacggatcagagagcccagagccaagctgcccgctcacaccaga ccctgctcagggtggcattgtctccccatggaaaaccagagaggagcactcagcctggtgtggtcactcttctcttatccactaaacggttgt cactgggcactgccaccagccccgtgtttctctgggtgtagggccctggggatgttacaggctgggggccaggtgacccaacactacag ggcaagatgagacaggcttccaggacacctagaatatcagaggaggtggcatttcaagcttttgtgattcattcgatgttaacattctttgactc aatgtagaagagctaaaagtagaacaaaccaaagccgagttcccatcttagtgtgggtggaggacacaggagtaagtggcagaaataatc agaaaagaaaacacttgcactgtggtgggtcccagaagaacaagaggaatgctgtgccatgccttgaatttcttttctgcacgacaggtctg ccagcttacatttacccaaactgtccattactggaacctatgatctgaagagcgtcctgggtcaactgggcatcactaaggtcttcagcaatg gggctgacctctccggggtcacagaggaggcacccctgaagctctccaaggtgagatcaccctgacgaccttgttgcaccctggtatctgt agggaagaatgtgtgggggctgcagctctgtcctgaggctgaggaaggggccgagggaaacaaatgaagacccaggctgagctcctg aagatgcccgtgattcactgacacgggacgtggtcaaacagcaaagccaggcaggggactgctgtgcagctggcactttcggggcctcc cttgaggttgtgtcactgaccctgaatttcaactttgcccaagaccttctagacattgggccttgatttatccatactgacacagaaaggtttggg ctaagttgtttcaaaggaatttctgactccttcgatctgtgagatttggtgtctgaattaatgaatgatttcagctaaagatgacacttattttggaa aactaaaggcgaccaatgaacaactgcagttccatgaatggctgcattatcttggggtctgggcactgtgaaggtcactgccagggtccgt gtcctcaaggagcttcaagccgtgtactagaaaggagagagccctggaggcagacgtggagtgacgatgctcttccctgttctgagttgtg ggtgcacctgagcagggggagaggcgcttgtcaggaagatggacagaggggagccagccccatcagccaaagccttgaggaggagc aaggcctatgtgacagggagggagaggatgtgcagggccagggccgtccagggggagtgagcgcttcctgggaggtgtccacgtgag ccttgctcgaggcctgggatcagccttacaacgtgtctctgcttctctcccctccaggccgtgcataaggctgtgctgaccatcgacAagaa agggactgaagctgctggggccatgtttttagaggccatacccatgtctatcccccccgaggtcaagttcaacaaaccctttgtcttcttaatg attgaacaaaataccaagtctcccctcttcatgggaaaagtggtgaatcccacccaaaaataactgcctctcgctcctcaacccctcccctcc atccctggccccctccctggatgacattaaagaagggttgagctggtccctgcctgcatgtgactgtaaatccctcccatgttttctctgagtct ccctttgcctgctgaggctgtatgtgggctccaggtaacagtgctgtcttcgggccccctgaactgtgttcatggagcatctggctgggtagg cacatgctgggcttgaatccaggggggactgaatcctcagcttacggacctgggcccatctgtttctggagggctccagtcttccttgtcctg tcttggagtccccaagaaggaatcacaggggaggaaccagataccagccatgaccccaggctccaccaagcatcttcatgtccccctgct catcccccactcccccccacccagagttgctcatcctgccagggctggctgtgcccaccccaaggctgccctcctgggggccccagaact gcctgatcgtgccgtggcccagttttgtggcatctgcagcaacacaagagagaggacaatgtcctcctcttgacccgctgtcacctaaccag actcgggccctgcacctctcaggcacttctggaaaatgactgaggcagattcttcctgaagcccattctccatggggcaacaaggacacct attctgtccttgtccttccatcgctgccccagaaagcctcacatatctccgtttagaatcaggtcccttctccccagatgaagaggagggtctct gctttgttttctctatctcctcctcagacttgaccaggcccagcaggccccagaagaccattaccctatatcccttctcctccctagtcacatgg ccataggcctgctgatggctcaggaaggccattgcaaggactcctcagctatgggagaggaagcacatcacccattgacccccgcaacc cctccctttcctcctctgagtcccgactggggccacatgcagcctgacttctttgtgcctgttgctgtccctgcagtcttcagagggccaccgc agctccagtgccacggcaggaggctgttcctgaatagcccctgtggtaagggccaggagagtccttccatcctccaaggccctgctaaag gacacagcagccaggaagtcccctgggcccctagctgaaggacagcctgctccctccgtctctaccaggaatggccttgtcctatggaag gcactgccccatcccaaactaatctaggaatcactgtctaaccactcactgtcatgaatgtgtacttaaaggatgaggttgagtcataccaaat agtgatttcgatagttcaaaatggtgaaattagcaattctacatgattcagtctaatcaatggataccgactgtttcccacacaagtctcctgttct cttaagcttactcactgacagcctttcactctccacaaatacattaaagatatggccatcaccaagccccctaggatgacaccagacctgaga gtctgaagacctggatccaagttctgacttttccccctgacagctgtgtgaccttcgtgaagtcgccaaacctctctgagccccagtcattgct agtaagacctgcctttgagttggtatgatgttcaagttagataacaaaatgtttatacccattagaacagagaataaatagaactacatttcttgc a

[0225] > All-in-one eNme2.C-TadA8e rAAV vector (SEQ ID NO: 8) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagt gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggcctctagaatggaggcggtactatgtagatg agaattcaggagcaaactgggaaaagcaactgcttccaaatatttgtgatttttacagtgtagttttggaaaaactcttagcctaccaattcttct aagtgttttaaaatgtgggagccagtacacatgaagttatagagtgttttaatgaggcttaaatatttaccgtaactatgaaatgctacgcatatc atgctgttcaggctccgtggccacgcaactcatactaccggtgccaccatgccaaagaagaagcggaaagtctctgaggtggagttttccc acgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagagggaggtgcctgtgggagccgtgctggtgctgaa caatagagtgatcggcgagggctggaacagagccatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggc ggcctggtcatgcagaactacagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatccact ctaggatcggccgcgtggtgtttggcgtgaggaactcaaaaagaggcgccgcaggctccctgatgaacgtgctgaactaccccggcatg aatcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaa tgctcagaagaaggcccagagctccatcaactctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgagtca gcaacaccagagagcagtggcggcagcagcggcggcagcgcagcattcaagtcaaacccaatcaattacatcctgggactggcaatcg gaatcgcatccgtgggatgggctatggtggagatcgacgaggaggggaatcctatccggctgatcgatctgggcgtgagagtgtttgaga gggccgaggtgccaaagaccggcgattctctggctatggcccggagactggcacggagcgtgaggcgcctgacacggagaagggca cacaggctgctgagggcacgccggctgctgaagagagagggcgtgctgcaggcagcagacttcgatgagaatggcctgatcacgagct tgccaaacaccccctggcagctgagagcagccgccctggacaggaagctgacaccactggagtggtctgccgtgctgctgcacctgatc aagcaccgcggctacctgagccagcggaagaacgagggagagacagcagccaaggagctgggcgccctgctgaagggagtggcca acaatgcccacgccctgcagaccggcgatttcaggacacctgccgagctggccctgaataagtttgagaaggagtccggccacatcaga aaccagaggggcgactatagccacaccttctcccgcaaggatctgcaggccgagctgatcctgctgttcgagaagcagaaggagtttggc aatccacacgtgagcggaggcctgaaggagggaatcgagaccctgctgatgacacagaggcctgccctgtccggcgacgcagtgcag aagatgctggggcactgcaccctcgagcctacagagccaaaggccgccaagaacacctacacagccgagcggtttatctggctgacaaa gctgaacaatctgagaatcctggagcagggatccgagaggccactgaccgacacagagaggtccaccctgatggatgagccttaccgg aagtctaaactgacatatgcccaggccagaaagctgctgggcctggaggacaccgccttctttaagggcctgagatacggcaaggataat gccgaggcctccacactgatggagatgaaggcctatcacgccatctctcgcgccctggagaaggagggcctgaaggacaagaagtccc ccctgaacctgagctccgagctgcaggatgagatcggcaccgccttctctctgtttaagaccgacgaggatatcacaggccgcctgaagg acagggtgcagcctgagatcctggaggccctgctgaagcacatctctttcgataagtttgtgcagatcagcctgaaggccctgagaaggat cgtgccactgatggagcagggcaagcggtacgacgaggcctgcgccgagatctacggcgttcactatggcaagaagaacacagagga gaagatctatctgccccctatccctgccgacgagatcagaaatcctgtggtgctgagggccctgtcccaggcaagaaaagtgatcaacgg agtggtgcgccggtacggatctccagcccggatccacatcgagaccgccagagaagtgggcaagagcttcaaggaccggaaggagat cgcgaagagacaggaggagaatcgcaaggatcgggagaaggccgccgccaagtttagggagtacttccctaactttgtgggcgagcca aagtctaaggacatcctgaagctgcgcctgtacgagcagcagcacggcaagtgtctgtatagcggcaaagagatcaatctggtgcggctg aacgagaagggctatgtggagatcgatcacgccctgcctttctccagaacctgggacgattcttttaacaataaggtgctggtgctgggcag cgagaaccagaataagggcaatcagacaccatacgagtatttcaatggcaaggacaactccagggagtggcaggagttcaaggcccgc gtggagacctctagatttcccagtagcaagaagcagcggatcctgctgcagaagttcgacgaggatggctttaaggagtgcaacctgaatg acaccagatacgtgaaccggttcctgtgccagtttgtggccgatcacatcctgctgaccggcaagggcaagagaagggtggtcgcctcta atggccagatcacaaacctgctgagggggttttggagactgaggaaggtgcgggcagagaatgacagacaccacgcactggatgcagt ggtggtggcatgcagcaccgtggcaatgcagcagaagatcacaagattcgtgaggtataaggagatgaacgcctttgacggcaagaccg tcgataaggagacaggcaaggtgctgtaccagaagacccacttcccccagccttgggagttctttgcccaggaagttatgatccgggtgttc ggcaagccagacggcaagcctgagtttgaggaggccgataccccagagaagctgaggacactgctggcagagaagctgtctagcagg ccagaggcagtgcacgagtacgtgaccccgctgttcgtgtccagggcacccaatcggaagatgtctggcgcccacaaggacacactga gaagcgccaagaggtttgtgaagcacaacgagaagatctccgtgaagagagtgtggctgaccgagatcaagctggccgatctggagaa catggtgaattacaagaacggcagggagatcgagctgtatgaggccctgaaggcaaggctggaggcctacggaggaaatgccaagca ggccttcgacccaaaggataaccccttttataagaagggaggacagctggtgaaggccgtgcgggtggagaagacccagaagagcggc gtgctgctgaataagaagaacgcctacacaatcgccgacaatggtgatatggtgagagtggacgtgttctgtaaggtggataagaagggc aagaatcagtactttatcgtgcctatctatgcctggcaggtggccgagaacatcctgccagacatcgattgcaagggctacagaatcgacga tagctatacattctgtttttccctgcacaagtatgacctgatcgccttccagaaggatgagaagtccaaggtggagtttgcctactatatcaattg cgactcctctagcggcgggttctacctggcctggcacgataagggcagcagggagcagcggtttcgcatctccacccagaatctggcgct gatccagaagtatcaggtgaacgagctgggcaaggagatcaggccatgtcggctgaagaagcgcccacccgtgcggtctggcggctca aaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtctaaaataaaggaaatttattttcattgcaatagtgtgttg gaattttttgtgtctctcagctagcGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGAT ACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTA GTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAA TTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCT TGGCTTTATATATCTTGTGGAAAGGACGAAACaccGccatcgacAagaaagggactgaaGTTGTA GCTCCCTTTCTCATTTCGGAAACGAAATGAGAACCGTTGCTACAATAAGGCCGTCTG AAAAGATGTGCCGCAACGCTCTGCCCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTT ATTTTTTTgcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgac caaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt attttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgg gtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgc cggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggt gatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact ggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaa aatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagcccc gacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagct gcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataat aatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctca tgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcgg cattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaact ggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggta ttatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaa gcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgat cggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagcc ataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttc ccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgata aatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgac ggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt actcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacg tgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcaga taccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtta ccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgcca cgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggg aaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgg aaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

[0226] > All-in-one eNme2.C-TadA9e rAAV vector (SEQ ID NO: 9) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagt gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggcctctagaatggaggcggtactatgtagatg agaattcaggagcaaactgggaaaagcaactgcttccaaatatttgtgatttttacagtgtagttttggaaaaactcttagcctaccaattcttct aagtgttttaaaatgtgggagccagtacacatgaagttatagagtgttttaatgaggcttaaatatttaccgtaactatgaaatgctacgcatatc atgctgttcaggctccgtggccacgcaactcatactaccggtgccaccatgccaaagaagaagcggaaagtctctgaggtggagttttccc acgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagagggaggtgcctgtgggagccgtgctggtgctgaa caatagagtgatcggcgagggctggaacagagccatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggc ggcctggtcatgcagaactacagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatccact ctaggatcggccgcgtggtgtttggcgtgaggaactcaaaaaCCggcgccgcaggctccctgatgaacgtgctgaactaccccggcat gaaGcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacGggtgtt caatgctcagaagaaggcccagagctccatcaactctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgag tcagcaacaccagagagcagtggcggcagcagcggcggcagcgcagcattcaagtcaaacccaatcaattacatcctgggactggcaa tcggaatcgcatccgtgggatgggctatggtggagatcgacgaggaggggaatcctatccggctgatcgatctgggcgtgagagtgtttg agagggccgaggtgccaaagaccggcgattctctggctatggcccggagactggcacggagcgtgaggcgcctgacacggagaagg gcacacaggctgctgagggcacgccggctgctgaagagagagggcgtgctgcaggcagcagacttcgatgagaatggcctgatcacg agcttgccaaacaccccctggcagctgagagcagccgccctggacaggaagctgacaccactggagtggtctgccgtgctgctgcacct gatcaagcaccgcggctacctgagccagcggaagaacgagggagagacagcagccaaggagctgggcgccctgctgaagggagtg gccaacaatgcccacgccctgcagaccggcgatttcaggacacctgccgagctggccctgaataagtttgagaaggagtccggccacat cagaaaccagaggggcgactatagccacaccttctcccgcaaggatctgcaggccgagctgatcctgctgttcgagaagcagaaggagt ttggcaatccacacgtgagcggaggcctgaaggagggaatcgagaccctgctgatgacacagaggcctgccctgtccggcgacgcagt gcagaagatgctggggcactgcaccctcgagcctacagagccaaaggccgccaagaacacctacacagccgagcggtttatctggctg acaaagctgaacaatctgagaatcctggagcagggatccgagaggccactgaccgacacagagaggtccaccctgatggatgagcctta ccggaagtctaaactgacatatgcccaggccagaaagctgctgggcctggaggacaccgccttctttaagggcctgagatacggcaagg ataatgccgaggcctccacactgatggagatgaaggcctatcacgccatctctcgcgccctggagaaggagggcctgaaggacaagaa gtcccccctgaacctgagctccgagctgcaggatgagatcggcaccgccttctctctgtttaagaccgacgaggatatcacaggccgcctg aaggacagggtgcagcctgagatcctggaggccctgctgaagcacatctctttcgataagtttgtgcagatcagcctgaaggccctgagaa ggatcgtgccactgatggagcagggcaagcggtacgacgaggcctgcgccgagatctacggcgttcactatggcaagaagaacacaga ggagaagatctatctgccccctatccctgccgacgagatcagaaatcctgtggtgctgagggccctgtcccaggcaagaaaagtgatcaa cggagtggtgcgccggtacggatctccagcccggatccacatcgagaccgccagagaagtgggcaagagcttcaaggaccggaagga gatcgcgaagagacaggaggagaatcgcaaggatcgggagaaggccgccgccaagtttagggagtacttccctaactttgtgggcgag ccaaagtctaaggacatcctgaagctgcgcctgtacgagcagcagcacggcaagtgtctgtatagcggcaaagagatcaatctggtgcgg ctgaacgagaagggctatgtggagatcgatcacgccctgcctttctccagaacctgggacgattcttttaacaataaggtgctggtgctggg cagcgagaaccagaataagggcaatcagacaccatacgagtatttcaatggcaaggacaactccagggagtggcaggagttcaaggcc cgcgtggagacctctagatttcccagtagcaagaagcagcggatcctgctgcagaagttcgacgaggatggctttaaggagtgcaacctg aatgacaccagatacgtgaaccggttcctgtgccagtttgtggccgatcacatcctgctgaccggcaagggcaagagaagggtggtcgcc tctaatggccagatcacaaacctgctgagggggttttggagactgaggaaggtgcgggcagagaatgacagacaccacgcactggatgc agtggtggtggcatgcagcaccgtggcaatgcagcagaagatcacaagattcgtgaggtataaggagatgaacgcctttgacggcaaga ccgtcgataaggagacaggcaaggtgctgtaccagaagacccacttcccccagccttgggagttctttgcccaggaagttatgatccgggt gttcggcaagccagacggcaagcctgagtttgaggaggccgataccccagagaagctgaggacactgctggcagagaagctgtctagc aggccagaggcagtgcacgagtacgtgaccccgctgttcgtgtccagggcacccaatcggaagatgtctggcgcccacaaggacacac tgagaagcgccaagaggtttgtgaagcacaacgagaagatctccgtgaagagagtgtggctgaccgagatcaagctggccgatctggag aacatggtgaattacaagaacggcagggagatcgagctgtatgaggccctgaaggcaaggctggaggcctacggaggaaatgccaagc aggccttcgacccaaaggataaccccttttataagaagggaggacagctggtgaaggccgtgcgggtggagaagacccagaagagcgg cgtgctgctgaataagaagaacgcctacacaatcgccgacaatggtgatatggtgagagtggacgtgttctgtaaggtggataagaaggg caagaatcagtactttatcgtgcctatctatgcctggcaggtggccgagaacatcctgccagacatcgattgcaagggctacagaatcgacg atagctatacattctgtttttccctgcacaagtatgacctgatcgccttccagaaggatgagaagtccaaggtggagtttgcctactatatcaatt gcgactcctctagcggcgggttctacctggcctggcacgataagggcagcagggagcagcggtttcgcatctccacccagaatctggcg ctgatccagaagtatcaggtgaacgagctgggcaaggagatcaggccatgtcggctgaagaagcgcccacccgtgcggtctggcggct caaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtctaaaataaaggaaatttattttcattgcaatagtgtgt tggaattttttgtgtctctcagctagcGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGA TACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAA ATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTC TTGGCTTTATATATCTTGTGGAAAGGACGAAACaccGccatcgacAagaaagggactgaaGTTGT AGCTCCCTTTCTCATTTCGGAAACGAAATGAGAACCGTTGCTACAATAAGGCCGTCT GAAAAGATGTGCCGCAACGCTCTGCCCCTTAAAGCTTCTGCTTTAAGGGGCATCGTT TATTTTTTTgcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcga ccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcg gtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcg ggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcg ccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttggg tgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaac tggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaa aaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccc cgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggag ctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgata ataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgct catgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcg gcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaac tggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggt attatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaa agcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacg atcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaag ccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagc ttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctga taaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg acggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccctta acgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgca aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgc agataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcct gttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcggg ctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagc gccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagccta tggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

[0227] EQUIVALENTS

[0228] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0229] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0230] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0231] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0232] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0233] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0234] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

Claims

CLAIMSWhat is claimed is:

1. An isolated nucleic acid comprising a transgene flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), the transgene comprising:(i) a first nucleic acid encoding an adenine deaminase;(ii) a second nucleic acid encoding an RNA-guided nuclease; and(iii) a third nucleic acid encoding a guide RNA (gRNA) that comprises a region of complementarity to a nucleic acid encoding a mutant alpha 1 antitrypsin (AAT) peptide.

2. The isolated nucleic acid of claim 1, wherein the adenine deaminase is a TadA-8e peptide.

3. The isolated nucleic acid of claim 2, wherein the TadA-8e peptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

4. The isolated nucleic acid of claim 1, wherein the adenine deaminase is a TadA-9e peptide.

5. The isolated nucleic acid of claim 4, wherein the TadA-9e peptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

6. The isolated nucleic acid of any one of claims 1 to 5, wherein the RNA-guided nuclease is a Neisseria meningitidis evolved Cas9 nickase (eNme2.C) peptide.

7. The isolated nucleic acid of claim 6, wherein the eNme2.C peptide comprises the amino acid sequence set forth in SEQ ID NO: 3.

8. The isolated nucleic acid of any one of claims 1 to 7, wherein the gRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 4-6.

9. The isolated nucleic acid of any one of claims 1 to 8, wherein the transgene further comprises one or more promoters.

10. The isolated nucleic acid of claim 9, wherein the transgene comprises a first promoter and a second promoter.

11. The isolated nucleic acid of claim 10, wherein the first promoter comprises a Ula promoter and the Ula promoter is operably linked to the first nucleic acid sequence.

12. The isolated nucleic acid of claim 10 or 11, wherein the second promoter comprises a U6 promoter and the U6 promoter is operably linked to the third nucleic acid sequence.

13. The isolated nucleic acid of any one of claims 1 to 12, wherein the transgene further comprises a linker molecule positioned between the first nucleic acid and the second nucleic acid.

14. The isolated nucleic acid of claim 13, wherein the linker molecule comprises an amino acid linker.

15. The isolated nucleic acid of any one of claims 1 to 14, wherein the mutant alpha 1 antitrypsin (AAT) peptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 7.

16. The isolated nucleic acid of any one of claims 1 to 15, wherein the AAV ITRs are AAV2 ITRs.

17. The isolated nucleic acid of any one of claims 1 to 16, comprising the nucleic acid sequence set forth in SEQ ID NO: 8 or 9.

18. A recombinant adeno-associated virus (rAAV) comprising:(i) the isolated nucleic acid of any one of claims 1 to 17; and(ii) one or more AAV capsid proteins.

19. The rAAV of claim 18, wherein the one or more AAV capsid proteins are AAV8 capsid proteins.

20. A method for reducing a mutant alpha 1 antitrypsin (AAT) peptide level in a subject in need thereof, the method comprising administering the isolated nucleic acid of any one of claims 1 to 17 or the rAAV of claim 18 or 19, to a subject comprising an alpha 1 antitrypsin (AAT) gene comprising one or more mutations.

21. The method of claim 20, wherein the one or more mutations comprise a G-to-A mutation that causes a glutamic acid to lysine amino acid change at position 342 of AAT.

22. The method of claim 20 or 21, wherein the subject is a mammal, optionally a human.

23. A method for base editing a nucleic acid encoding a mutant alpha 1 antitrypsin (AAT) peptide, the method comprising contacting a cell comprising the nucleic acid with the isolated nucleic acid of any one of claims 1 to 17 or the rAAV of claim 18 or 19.

24. The method of claim 23, wherein the cell is a mammalian cell, optionally a human cell.

25. A method for treating a subject having alpha 1 antitrypsin (AAT) deficiency, the method comprising administering to a subject in need thereof the isolated nucleic acid of any one of claims 1 to 17 or the rAAV of claim 18 or 19.

26. The method of claim 25, wherein the subject is a mammal, optionally a human.

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