AAV gene therapy for alpha-1 antitrypsin deficiency

Abstract

The present disclosure relates to gene therapy for treating Alpha-1 antitrypsin deficiency (A1AD). Provided here are multifunctional AAV vectors that simultaneously deliver an RNAi molecule targeting a mutant AAT allele (e.g., the PiZ allele) and a normal AAT gene (e.g., the PiM allele). Also provided here are variant bovine AAV (BAAV) capsid proteins and variant capsid protein containing particles with enhanced ability to transduce macrophages.

Description

[0001] AAV GENE THERAPY FOR ALPHA-1 ANTITRYPSIN DEFICIENCY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 517,752, filed August 4, 2023, entitled “AAV GENE THERAPY FOR ALPHA- 1 ANTITRYPSIN DEFICIENCY,” the entire content of which is incorporated herein by reference.

[0004] BACKGROUND

[0005] Alpha-1 antitrypsin deficiency (A1AD) is the most common rare disease discovered in humans thus far. It is estimated that 5% of the population suffer from A1AD. In 2022, the estimated human population was approximated to be 8 billion people, with around 4 million individuals suffering from A1AD across the globe. In the United States alone, about 100,000 suffer from A1AD. Throughout the COVID19 pandemic, individuals with A1AD comprised a sizable portion of life lost. The effects of A1AD on the lungs predisposed many individuals to more severe adverse events.

[0006] As of 2018, the cost of treatment for A1AD was estimated to be around $127,000 per year. The treatment is derived from human plasma donation from which functional alpha- 1 antitrypsin (AAT) is purified. Augmentation therapy is very costly to produce, with weekly infusions worsening the financial burden on the patient. Using human plasma as the source for AAT also increases the risk of patients to other pathogens every time the augmentation treatment is administered.

[0007] As of today, 150 mutations have been discovered within the AAT gene, but only one, the PiZ mutation, causes the most debilitating symptoms. The PiZ mutation is a single nucleotide change (C to T) that results in an amino acid substitution - glutamate to lysine at position 342 of the AAT. This single amino acid change induces a conformational change in the AAT protein that causes polymerization of the protein. Polymerization of PiZ will create aggregates inside the cells and induce the following: a significant reduction of secretion of AAT into the plasma and accumulation of PiZ in the cells (liver and mononuclear phagocytes). Lacking AAT in the blood and local tissues predisposes the organs to injury induced by serine proteases, oxidants and neutrophil defensins. For example, this damage in the lungs leads to chronic obstructive pulmonary disease (COPD). Macrophages in PiZ individuals have impaired key functions including excess production of pro-inflammatory factors and impair efferocytosis. Hepatocytes are the major producer of AAT in the body. PiZ individuals develop issues both because of accumulation of PiZ-AAT in cells and the lack of AAT to regulate inflammation and tissue degradation. Accumulation of mutant Z-AAT protein in the liver and lung can cause cirrhosis, HCC, and pulmonary function impairment.

[0008] Thus, there is a need to develop an effective and affordable gene therapy for the treatment of Al AD.

[0009] SUMMARY

[0010] In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a recombinant nucleic acid comprising (i) inverted terminal repeats (ITRs); (ii) a nucleotide sequence encoding an siRNA targeting the PiZ allele of AAT, wherein the siRNA comprises the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16; and (iii) a nucleotide sequence encoding PiM-AAT. In some embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a recombinant nucleic acid comprising (i) inverted terminal repeats (ITRs); (ii) a nucleotide sequence encoding an siRNA targeting the PiZ allele of AAT, wherein the siRNA comprises the sequence set forth in SEQ ID NO: 10; and (iii) a nucleotide sequence encoding PiM- AAT. In some embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a recombinant nucleic acid comprising (i) inverted terminal repeats (ITRs); (ii) a nucleotide sequence encoding an siRNA targeting the PiZ allele of AAT, wherein the siRNA comprises the sequence set forth in SEQ ID NO: 16; and (iii) a nucleotide sequence encoding PiM-AAT.

[0011] In some embodiments, the recombinant nucleic acid comprises, from 5’ to 3’, a 5’ ITR, an enhancer, a promoter, the nucleotide sequence encoding the siRNA inserted into an intron, the nucleotide sequence encoding PiM-AAT, and a 3’ ITR. In some embodiments, the intron is the chicken beta-actin intron donor of the CAG promoter. In some embodiments, the intron is the SV40 intron donor.

[0012] In some embodiments, the recombinant nucleic acid comprises a liver specific promoter operably linked to the nucleotide sequence encoding the siRNA and / or the nucleotide sequence encoding PiM-AAT. In some embodiments, the recombinant nucleic acid comprises a lung specific promoter operably linked to the nucleotide sequence encoding the siRNA and / or the nucleotide sequence encoding PiM-AAT. In some embodiments, the recombinant nucleic acid comprises a macrophage specific promoter operably linked to the nucleotide sequence encoding the siRNA and / or the nucleotide sequence encoding PiM- AAT. In some embodiments, the recombinant nucleic acid is self-complementary.

[0013] In one aspect, the present disclosure provides an rAAV particle comprising an rAAV vector of the disclosure. In some embodiments, the rAAV particle targets liver cells. In some embodiments, the rAAV particle targets lung cells. In some embodiments, the rAAV particle targets macrophages. In some embodiments, the rAAV particle is of serotype AAV3. In some embodiments, the rAAV particle has a bovine AAV (BAAV) capsid.

[0014] In one aspect, the present disclosure provides a composition comprising a plurality of rAAV particles of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0015] In one aspect, the present disclosure provides a method of administering an rAAV particle of the disclosure or a composition comprising a plurality of rAAV particles of the disclosure to a subject.

[0016] In one aspect, the present disclosure provides a method of treating A1AD in a subject in need thereof, comprising administering to the subject an rAAV particle of the disclosure or a composition of the disclosure. In some embodiments, the subject is human.

[0017] In one aspect, the disclosure provides a variant bovine adeno-associated virus (BAAV) capsid protein comprising one or more of the following insertions in SEQ ID NO: 25: (i) a VAAA-X (SEQ ID NO: 32) insertion at position 45 of SEQ ID NO: 25, wherein X is L or S; and (ii) a GAAA-X (SEQ ID NO: 33) insertion at position 15 of SEQ ID NO: 25, wherein X is A or T. In some embodiments, the variant BAAV capsid protein comprises a VAAAL (SEQ ID NO: 34) insertion at position 45 of SEQ ID NO: 25. In some embodiments, the variant BAAV capsid protein comprises a VAAAS (SEQ ID NO: 35) insertion at position 45 of SEQ ID NO: 25. In some embodiments, the variant BAAV capsid protein comprises a GAAAA (SEQ ID NO: 36) insertion at position 15 of SEQ ID NO: 25. In some embodiments, the variant BAAV capsid protein comprises a GAAAT (SEQ ID NO: 37) insertion at position 15 of SEQ ID NO: 25.

[0018] In one aspect, the present disclosure provides a recombinant BAAV (rBAAV) particle comprising a variant BAAV capsid protein of the disclosure. In some embodiments, the rBAAV particle has enhanced tropism for a macrophage compared to an rBAAV particle comprising a capsid protein having the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the rBAAV has enhanced transduction efficiency for a macrophage compared to an rBAAV particle comprising a capsid protein having the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the rBAAV particle further comprises a recombinant nucleic acid comprising inverted terminal repeats (ITRs). In some embodiments, the recombinant nucleic acid of the rBAAV particle comprises a nucleotide sequence encoding an siRNA targeting the PiZ allele of AAT. In some embodiments, the siRNA targeting the PiZ allele of AAT comprises the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid further comprises a nucleotide sequence encoding PiM-AAT. In some embodiments, the recombinant nucleic acid further comprises a macrophage specific promoter. In some embodiments, the recombinant nucleic acid comprises, from 5’ to 3’, a 5’ ITR, an enhancer, a promoter, the nucleotide sequence encoding the siRNA inserted into an intron, the nucleotide sequence encoding PiM-AAT, and a 3’ ITR. In some embodiments, the recombinant nucleic acid is self-complementary.

[0019] In one aspect, the present disclosure provides a composition comprising a plurality of the rBAAV particles of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0020] In one aspect, the present disclosure provides a method of administering an rBAAV particle of the disclosure or a composition comprising an rBAAV particle of the disclosure to a subject.

[0021] In one aspect, the present disclosure provides a method of transducing a macrophage, the method comprising contacting the macrophage with an rBAAV particle of the disclosure or a composition comprising an rBAAV particle of the disclosure.

[0022] In one aspect, the present disclosure provides a method of treating Alpha- 1 antitrypsin deficiency (A1AD) in a subject in need thereof, comprising administering to the subject an rBAAV particle of the disclosure or a composition comprising an rBAAV particle of the disclosure. In some embodiments, the subject is human.

[0023] In one aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 20 is operably linked to (ii) a nucleotide sequence encoding an siRNA targeting the PiZ allele of AAT, wherein the siRNA comprises the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16; and / or (iii) a nucleotide sequence encoding PiM-AAT.

[0024] In one aspect, the present disclosure provides a vector comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In one aspect, the present disclosure provides an rAAV particle comprising the rAAV vector encapsidated in an AAV capsid. In some embodiments, the AAV capsid comprises a capsid protein derived from bovine AAV (BAAV). In one aspect, the present disclosure provides a composition comprising the rAAV particle.

[0025] In one aspect, the present disclosure provides a method of treating A1AD in a subject in need thereof, comprising administering to the subject an isolated nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 20, an rAAV particle comprising the nucleic acid, or a composition comprising the rAAV particle.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

[0028] FIG. 1 shows a schematic of a multifunctional gene therapy vector. An RNAi molecule and a gene of interest are simultaneously delivered via an AAV vector. For example, an siRNA molecule targeting the mutant PiZ allele of alpha- 1 antitrypsin (AAT) and the gene for the normal PiM allele of the AAT gene are simultaneously delivered via an AAV vector.

[0029] FIG. 2 shows a dsAAV encoding M-AAT as well as the PiZ-specific siRNA, PIO. The siRNA is inserted into an intron.

[0030] FIGs. 3A and 3B show the schematic for CAG promoter modification and SV40 intron modification, respectively, with the plO siRNA.

[0031] FIGs. 4A and 4B show AAV plasmids for simultaneous PiZ-specific silencing and M-AAT augmentation. FIG. 4A shows a plasmid wherein the plO siRNA has been inserted into the CAG promoter intron and FIG. 4B shows a plasmid wherein the p 10 siRNA has been inserted into the SV40 intron.

[0032] FIG. 5A shows allele- specific siRNA molecules that can preferentially target the PiZ mutant AAT over the PiM AAT. The sequences correspond to SEQ ID NOs. 1-19, from top to bottom. PIO corresponds to SEQ ID NO: 10 and P16 corresponds to SEQ ID NO: 16. FIG. 5B shows the sequence of p 10 as a hairpin RNA. The top sequence corresponds to SEQ ID NO: 29. The bottom sequence corresponds to SEQ ID NO: 27. FIG. 6 shows a PiZ specific siRNA (plO) AAV plasmid.

[0033] FIG. 7 shows percent knockdown of AAT as compared to cells transfected with a negative control (NC) shRNA in in vitro studies.

[0034] FIG. 8 shows IHC staining for human AAT from the livers of human PiZ transgenic mice after portal vein delivery of rAAV8-U6-plO shRNA or negative control (NC) vector.

[0035] FIG. 9 shows total serum AAT levels in PiZ mice after portal vein injections with allele specific plO shRNA or negative control packaged in rAAV8.

[0036] FIG. 10 shows an exemplary AAV plasmid for simultaneous PiZ-specific silencing and M-AAT augmentation.

[0037] FIG. 11 shows the structures of plO-miRNA and plO-siRNA.

[0038] FIG. 12 shows the secretion level of AAT in cell supernatants after treatment with UF11 (negative control), 3X (positive control), plO-miRNA, and plO-siRNA.

[0039] FIG. 13 shows the AAT levels in cell pellets after treatment with UF11 (negative control), 3X (positive control), plO-miRNA, and plO-siRNA.

[0040] FIGs. 14A-14B show the replication of different viruses in mouse and human macrophages. FIG. 14A shows replication of different recombinant AAV-CBA-tdTomato particles in mouse macrophages at day 12 after transduction. FIG. 14B shows replication of different recombinant dsAAV-CD68-MVMi-mCherry particles in mouse macrophages. FIG. 14C shows replication of different recombinant AAV-CD33-EGFP particles in mouse macrophages. FIG. 14D shows replication of different recombinant AAVs in human monocyte-derived macrophages at day 8 after transduction.

[0041] FIG. 15 shows the flowchart of the protocol for the transposon-based mutation generation system (MGS) system.

[0042] FIG. 16 shows an exemplary dsAAV plasmid for simultaneous PiZ-specific silencing and M-AAT augmentation comprising a mini CMV promoter and SV40 intron modification.

[0043] FIG. 17 shows an exemplary dsAAV plasmid for simultaneous PiZ-specific silencing and M-AAT augmentation comprising a hCD68 promoter and SV40 intron modification.

[0044] FIG. 18 shows an exemplary dsAAV plasmid encoding GFP and comprising an hCD68 promoter and an MVM intron.

[0045] FIG. 19 shows an exemplary dsAAV plasmid encoding GFP and comprising a mini CMV promoter and an SV40 intron modified with plO siRNA.

[0046] FIG. 20 shows an exemplary plasmid encoding a variant bovine AAV capsid protein.

[0047] FIG. 21 shows the nucleotide sequence of a truncated human CD68 promoter along with a restriction site map. FIG. 22 shows an exemplary AAV plasmid encoding RFP and comprising the truncated hCD68 promoter.

[0048] FIG. 23 shows the results of GFP and RFP fluorescence imaging in mice brain slices.

[0049] FIG. 24 shows the results of GFP fluorescence imaging in HEK293T cells, primary neuroglia culture, brain slice culture, and spinal cord slice culture.

[0050] DETAILED DESCRIPTION

[0051] The following detailed description is made by way of illustration of certain aspects of the disclosure. It is to be understood that other aspects are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense. Scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0052] The present disclosure relates to compositions and methods of gene therapy for treating A1AD, e.g., A1AD caused by the PiZ mutation. In some embodiments, the compositions and methods of the disclosure target the two main organs that are more affected by PiZ-affected A1AD: lung and liver. The compositions and methods of the present disclosure target the single nucleotide mutation of PiZ and leave the normal PiM allele (M- AAT) unaffected by RNA silencing. In one aspect, the present disclosure provides a single gene therapy vector (e.g., an AAV vector) that is capable of simultaneously delivering two different RNA molecules for the treatment of Al AD. Thus, in some embodiments, the present disclosure provides a gene therapy vector that comprises a first RNA molecule: a PiZ- specific gene silencing interfering RNA (RNAi) (e.g., miRNA, siRNA, or shRNA), to silence the PiZ allele; and a second RNA molecule: the PiM mRNA, to upregulate expression of healthy AAT. In another aspect, the present disclosure provides RNAi molecules for specifically targeting the PiZ allele of AAT. In yet another aspect, the present disclosure provides variant bovine AAV capsid proteins that have enhanced tropism and / or transduction efficiency for macrophages (e.g., lung macrophages).

[0053] In some embodiments, the two RNA molecules are delivered using a self- complementary adeno associated virus (scAAV or dsAAV) vector. scAAV allows for higher in vivo DNA stability and more effective circulation of the vector genome. Rapid and longterm RNA transcription results in higher gene expression is another advantage of scAAV when compared to single- strand AAV vectors. One challenge of using sc AAV vectors is the limited packaging capacity. Since the vector is double stranded, the therapeutic cargo capacity of the virus is cut in half (about 2500 nt). To overcome this barrier, several short gene expression cassettes have been developed that fit within the limited packaging capacity. Thus, in one aspect, the present disclosure provides short gene expression cassettes (< about 3 kb) that encode a PiZ-specific gene silencing interfering RNA, and the PiM mRNA. In another aspect, the present disclosure provides a truncated macrophage-specific promoter.

[0054] Allele-specific RNAi molecules

[0055] The present disclosure provides RNAi molecules that specifically target the PiZ allele of AAT. Such RNAi molecules can be delivered via rAAV particles to specifically silence the mutant PiZ allele, which leaving the normal PiM allele intact. In some embodiments, the RNAi molecule is an siRNA molecule that comprises a sequence set forth in any one of SEQ ID NOs. 1-19. In some embodiments, the RNAi molecule is an siRNA molecule that comprises a sequence of the complement of any one of SEQ ID NOs. 1-19. In some embodiments, an RNAi molecule of the disclosure comprises a sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs. 1-19. In some embodiments, the RNAi molecule is an siRNA molecule that comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the RNAi molecule is an siRNA molecule that consists of the sequence set forth in SEQ ID NO: 10. In some embodiments, an RNAi molecule of the disclosure comprises a sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10. In some embodiments, the RNAi molecule is an siRNA molecule comprising the sequence set forth in SEQ ID NO: 27. In some embodiments, the RNAi molecule is an siRNA molecule that consists of the sequence set forth in SEQ ID NO: 27. In some embodiments, an RNAi molecule of the disclosure comprises a sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 27. In some embodiments, the RNAi molecule is an siRNA molecule that comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the RNAi molecule is an siRNA molecule that consists of the sequence set forth in SEQ ID NO: 16. In some embodiments, an RNAi molecule of the disclosure comprises a sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 16.

[0056] Multifunctional AAV vectors

[0057] The present disclosure provides multifunctional AAV vectors that are capable of simultaneously delivering an RNAi molecule that targets a mutant allele of AAT (e.g., PiZ) and the gene for the wild-type allele (PiM). In some embodiments, a multifunctional AAV vector comprises a nucleotide sequence encoding a siRNA that targets PiZ and a nucleotide sequence that encodes a wild-type AAT (PiM). In some aspects, a nucleotide sequence that encodes a wild-type AAT (PiM) comprises a nucleic acid of SEQ ID NO: 67. In some embodiments, the present disclosure provides an rAAV particle comprising a rAAV vector of the disclosure encapsidated in an AAV capsid.

[0058] As used herein, “AAV” is adeno-associated virus, and may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise. As used herein, the term “serotype” refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, and BAAV. For example, serotype AAV6 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV6 and a genome containing 5' and 3' ITR sequences from the same AAV6 serotype. Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs of a second serotype. Pseudotyped rAAV would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the second serotype. The abbreviation “rAAV” refers to recombinant adeno- associated viral particle or a recombinant AAV vector (or "rAAV vector"). An “AAV virus” or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as “rAAV.”

[0059] In some embodiments, an rAAV vector of the disclosure comprises a recombinant nucleic acid comprising (i) inverted terminal repeats (ITRs); (ii) a nucleotide sequence encoding an RNAi molecule that specifically targets the PiZ allele of AAT; and (iii) a nucleotide sequence encoding PiM-AAT. The ITR sequences may be wild-type ITR sequences or engineered ITR sequences. The ITR sequences flank the heterologous nucleic acid region (i.e., the sequences encoding the RNAi molecule and the sequence encoding PiM- AAT).

[0060] The application also provides the complement of any sequence described herein. Nucleic Acids

[0061] In some embodiments, a recombinant nucleic acid comprises one or more nucleic acid regions comprising a control sequence that facilitates expression of the heterologous nucleic acid region (e.g., a promoter) and a nucleotide sequence encoding a heterologous gene, e.g., a PiM- AAT. In some embodiments, a nucleic acid encoding a PiM-AAT comprises the sequence of SEQ ID NO: 67. In some embodiments, a nucleic acid encoding a PiM-AAT comprises the complement of a sequence of SEQ ID NO: 67. In some embodiments, an rAAV vector comprises one or more nucleic acid regions comprising a sequence that facilitates integration of the heterologous nucleic acid region (optionally with the one or more nucleic acid regions comprising a sequence that facilitates expression) into the genome of the subject.

[0062] Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer).

[0063] In some embodiments, one or more promoters may be operably linked to the nucleotide sequence encoding an RNAi molecule that specifically targets the PiZ allele of AAT and / or the nucleotide sequence encoding PiM-AAT. A promoter is “operably linked” to a nucleotide sequence when the promoter sequence controls and / or regulates the transcription of the nucleotide sequence. A promoter may be a constitutive promoter, tissuespecific promoter, an inducible promoter, or a synthetic promoter.

[0064] For example, constitutive promoters of different strengths can be used. A nucleic acid vector described herein may include one or more constitutive promoters, such as viral promoters or promoters from mammalian genes that are generally active in promoting transcription. Non-limiting examples of constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A cytomegalovirus (CMV) promoters. Non-limiting examples of constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the P-actin promoter (e.g., chicken P-actin promoter) and human elongation factor- 1 a (EF-la) promoter. In some embodiments, chimeric viral / mammalian promoters may include a chimeric CMV / chicken beta actin (CBA, CB or CAG) promoters.

[0065] Inducible promoters and / or regulatory elements may also be contemplated for achieving appropriate expression levels of the protein or polypeptide of interest. Nonlimiting examples of suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone- inducible genes, such as the estrogen gene promoter. Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline.

[0066] Tissue- specific and cell-specific promoters and / or regulatory elements are also contemplated herein. A “tissue- specific promoter”, as used herein, refers to promoters that preferentially or selectively function in a specific type of tissue. In some embodiments, a tissue-specific promoter is not able to drive the expression of the genes in other types of tissues. A “cell- specific promoter”, as used herein, refers to promoters that preferentially or selectively function in a specific type of cell. In some embodiments, a cell-specific promoter is not able to drive the expression of the genes in other types of cells.

[0067] In some embodiments, a promoter that may be used in accordance with the present disclosure is a liver- specific promoter. In some embodiments, a promoter that may be used in accordance with the present disclosure is a lung-specific promoter. In some embodiments, a promoter is a macrophage specific promoter.

[0068] In some embodiments, a promoter comprises a CMVdl promoter of SEQ ID NO: 64. In some embodiments, a promoter comprises a complement of SEQ ID NO: 64. In some embodiments, a promoter comprises a U6 promoter of SEQ ID NO: 81. In some embodiments, a promoter comprises a complement of SEQ ID NO: 81.

[0069] In some embodiments, a macrophage- specific promoter of the disclosure comprises a nucleotide sequence that is 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 NO: 64. In some embodiments, a macrophage- specific promoter of the disclosure consists essentially of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 64. In some embodiments, a macrophage-specific promoter of the disclosure consists of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 64. In some embodiments, a macrophage-specific promoter comprises a nucleotide sequence that has about 1-20, e.g., 1-5, 6-10, 11-15, or 16-20 nucleotide substitutions compared to a sequence of SEQ ID NO: 64.

[0070] In some embodiments, a macrophage- specific promoter comprises the nucleotide sequence of SEQ ID NO: 64. In some embodiments, a macrophage- specific promoter consists essentially of the nucleotide sequence of SEQ ID NO: 64. In some embodiments, a macrophage- specific promoter consists of the nucleotide sequence of SEQ ID NO: 64.

[0071] In some embodiments, a macrophage- specific promoter of the disclosure is a human CD68 promoter. In some embodiments, a human CD68 promoter comprises more than about 550 bp. In some embodiments, a human CD68 promoter comprises more than about 500 bp. In some embodiments, a human CD68 promoter comprises more than about 450 bp. In some embodiments, a human CD68 promoter comprises more than about 400 bp. In some embodiments, a human CD68 promoter comprises more than about 350 bp. In some embodiments, a promoter comprises a human CD68 promoter of SEQ ID NO: 73. In some embodiments, a macrophage-specific promoter of the disclosure comprises a nucleotide sequence that is 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 NO: 73. In some embodiments, a macrophage- specific promoter of the disclosure consists essentially of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 73. In some embodiments, a macrophage-specific promoter of the disclosure consists of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 73. In some embodiments, a macrophage-specific promoter comprises a nucleotide sequence that has about 1-20, e.g., 1-5, 6-10, 11-15, or 16-20 nucleotide substitutions compared to a sequence of SEQ ID NO: 73. In some embodiments, a macrophage- specific promoter comprises the nucleotide sequence of SEQ ID NO: 73. In some embodiments, a macrophage- specific promoter consists essentially of the nucleotide sequence of SEQ ID NO: 73. In some embodiments, a macrophage-specific promoter consists of the nucleotide sequence of SEQ ID NO: 73. In some embodiments, a macrophage- specific promoter comprises a complement of a nucleotide sequence of SEQ ID NO: 73.

[0072] Synthetic promoters are also contemplated herein. A synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like.

[0073] It is to be understood that a promoter may be a fragment of any one of the promoters disclosed herein, or one that retains partial promoter activity (e.g., 10-90, 30-60, 50-80,80-99 or 90-99.9% of the activity) of a whole promoter.

[0074] Any nucleic acid vector described herein may be encapsidated by a viral capsid. In some embodiments, the nucleic acid vector is circular. In some embodiments, the nucleic acid vector is single- stranded. In some embodiments, the nucleic acid vector is double- stranded. In some embodiments, a double-stranded nucleic acid vector may be, for example, a self- complimentary vector that contains a region of the nucleic acid vector that is complementary to another region of the nucleic acid vector, initiating the formation of the double- strandedness of the nucleic acid vector.

[0075] Truncated, promoters

[0076] In one aspect, the present disclosure provides a truncated macrophage- specific promoter. Truncated promoters enable the generation of shorter expression cassettes for use in dsAAV vectors. In some embodiments, the promoter is a truncated human CD68 (hCD68) promoter. In some embodiments, a truncated macrophage- specific promoter of the disclosure is less than about 500 bp in length. In some embodiments, a truncated macrophage- specific promoter is less than about 450 bp in length. In some embodiments, a truncated macrophagespecific promoter is less than about 400 bp in length. In some embodiments, a truncated macrophage- specific promoter is less than about 350 bp in length. In some embodiments, a truncated macrophage- specific promoter is less than about 300 bp in length. In some embodiments, a truncated macrophage- specific promoter is less than about 250 bp in length. In some embodiments, a truncated macrophage- specific promoter is less than about 200 bp in length. In some embodiments, the skeletal muscle- specific promoter is less than about 350 bp in length.

[0077] In some embodiments, a truncated macrophage- specific promoter of the disclosure comprises a nucleotide sequence that is 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 NO: 20. In some embodiments, a truncated macrophage- specific promoter of the disclosure consists essentially of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 20. In some embodiments, a truncated macrophage- specific promoter of the disclosure consists of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 20. In some embodiments, a truncated macrophage- specific promoter comprises the nucleotide sequence of SEQ ID NO: 20. In some embodiments, a truncated macrophagespecific promoter consists essentially of the nucleotide sequence of SEQ ID NO: 20. In some embodiments, a truncated macrophage- specific promoter consists of the nucleotide sequence of SEQ ID NO: 20. In some embodiments, a truncated macrophage-specific promoter comprises a nucleotide sequence that is the complement of SEQ ID NO: 20. In some embodiments, a truncated macrophage- specific promoter of the disclosure comprises a nucleotide sequence that is 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 NO: 65. In some embodiments, a truncated macrophage- specific promoter of the disclosure consists essentially of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 65. In some embodiments, a truncated macrophage- specific promoter of the disclosure consists of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 65. In some embodiments, a truncated macrophage- specific promoter comprises a nucleotide sequence that has about 1-20, e.g., 1-5, 6-10, 11-15, or 16-20 nucleotide substitutions compared to a sequence of SEQ ID NO: 65. In some embodiments, a truncated macrophage- specific promoter comprises the nucleotide sequence of SEQ ID NO: 65. In some embodiments, a truncated macrophage- specific promoter consists essentially of the nucleotide sequence of SEQ ID NO: 65. In some embodiments, a truncated macrophagespecific promoter consists of the nucleotide sequence of SEQ ID NO: 65. In some embodiments, a truncated macrophage- specific promoter comprises a nucleotide sequence that is the complement of SEQ ID NO: 65.

[0078] In some embodiments, a truncated macrophage- specific promoter of the disclosure comprises a nucleotide sequence that is 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 NO: 74. In some embodiments, a truncated macrophage- specific promoter of the disclosure consists essentially of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 74. In some embodiments, a truncated macrophage- specific promoter of the disclosure consists of a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 74. In some embodiments, a truncated macrophage- specific promoter comprises a nucleotide sequence that has about 1-20, e.g., 1-5, 6-10, 11-15, or 16-20 nucleotide substitutions compared to a sequence of SEQ ID NO: 74. In some embodiments, a truncated macrophage- specific promoter comprises the nucleotide sequence of SEQ ID NO: 74. In some embodiments, a truncated macrophage- specific promoter consists essentially of the nucleotide sequence of SEQ ID NO: 74. In some embodiments, a truncated macrophagespecific promoter consists of the nucleotide sequence of SEQ ID NO: 74. In some embodiments, a truncated macrophage- specific promoter comprises a nucleotide sequence that is the complement of SEQ ID NO: 74.A truncated macrophage-specific promoter may be used to express any gene of interest in a macrophage. Thus, a truncated macrophage-specific promoter of the disclosure may be operably linked to any gene of interest, to express the gene of interest in a macrophage. In some embodiments, the gene of interest is an RNAi molecule of the disclosure.

[0079] In some aspects, a truncated promoter comprising SEQ ID NO: 73 is operably linked to a gene of interest, e.g., a PiM-AAT gene. In some aspects, a truncated promoter comprising SEQ ID NO: 20 is operably linked to a gene of interest, e.g., a PiM-AAT gene. In some aspects, a truncated promoter comprising SEQ ID NO: 65 is operably linked to a gene of interest, e.g., a PiM-AAT gene. In some aspects, a truncated promoter comprising SEQ ID NO: 74 is operably linked to a gene of interest, e.g., a PiM-AAT gene. A truncated macrophage- specific promoter of the disclosure may be used for gene therapy for a disease or disorder associated with macrophages. In some embodiments, a truncated macrophagespecific promoter of the disclosure may be used for gene therapy for A1AD.

[0080] Introns

[0081] In some embodiments, a nucleic acid described herein comprises a nucleotide sequence encoding a siRNA that is inserted into an intron sequence. In some embodiments, a nucleotide sequence encoding a siRNA is present adjacent to an intron sequence. In some embodiments, a nucleic acid comprises an intron sequence comprising a siRNA and an allele of a gene of interest, e.g., a PiM-AAT allele operably linked to a promoter, e.g., a human CD68 promoter. Advantageously, by using an intron to encode the RNAi molecule, the mRNA encoding the normal allele undergoes normal processing and elicits long-term protein expression.

[0082] In some embodiments, a nucleic acid comprises an intron comprising a chicken betaactin intron donor of a chimeric CMV / chicken beta actin (CAG) promoter. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 78. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 78. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises an intron that consists essentially of a nucleotide sequence that is 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 NO: 78. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises an intron that consists of a nucleotide sequence that is 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 NO: 78. In some embodiments, an intron comprises a nucleotide sequence that has about 1-20, e.g., 1-5, 6-10, 11-15, or 16-20 nucleotide substitutions compared to a sequence of SEQ ID NO: 78. In some embodiments, an intron comprises the nucleotide sequence of SEQ ID NO: 78. In some embodiments, an intron consists essentially of the nucleotide sequence of SEQ ID NO: 78. In some embodiments, an intron consists of the nucleotide sequence of SEQ ID NO: 78. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 78.

[0083] In some embodiments, an intron comprises a nucleotide sequence of SEQ ID NO: 79. In some embodiments, an intron comprises a nucleotide sequence that is 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 NO: 79 In some embodiments, an intron consists essentially of a nucleotide sequence that is 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 NO: 79. In some embodiments, an intron consists of a nucleotide sequence that is 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 NO: 79. In some embodiments, an intron comprises a nucleotide sequence that has about 1-20, e.g., 1-5, 6-10, 11-15, or 16- 20 nucleotide substitutions compared to a sequence of SEQ ID NO: 79. In some embodiments, an intron consists essentially of the nucleotide sequence of SEQ ID NO: 79. In some embodiments, an intron consists of the nucleotide sequence of SEQ ID NO: 79. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 79.

[0084] In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 80. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 80. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that consists essentially of a nucleotide sequence that is 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 NO: 80. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence that is 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 NO: 80. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 80.

[0085] In some embodiments, a nucleotide sequence has about 1-10, e.g., 1-5, or 6-10 nucleotide substitutions compared to a sequence of SEQ ID NO: 80. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA consists essentially of a nucleotide sequence of SEQ ID NO: 80. In some embodiments, a nucleic acid sequence comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence of SEQ ID NO: 80.

[0086] In some embodiments, a nucleotide sequence encoding a siRNA is present in a SV40 intron. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 66. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 66. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of a nucleotide sequence that is 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 NO: 66. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence that is 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 NO: 66. In some embodiments, a nucleotide sequence has about 1-10, e.g., 1-5, or 6-10 nucleotide substitutions compared to a sequence of SEQ ID NO: 66. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of the nucleotide sequence of SEQ ID NO: 66. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises consists of the nucleotide sequence of SEQ ID NO: 66. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 66.

[0087] In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA is present adjacent to a SV40 intron sequence of SEQ ID NO: 68. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA is present adjacent to a SV40 intron sequence of SEQ ID NO: 69. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 70. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 70. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of a nucleotide sequence that is 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 NO: 70. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence that is 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 NO: 70. In some embodiments, a nucleotide sequence has about 1-10, e.g., 1-5, or 6-10 nucleotide substitutions compared to a sequence of SEQ ID NO: 70. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of the nucleotide sequence of SEQ ID NO: 70. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of the nucleotide sequence of SEQ ID NO: 70. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 70.

[0088] In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 71. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 71. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of a nucleotide sequence that is 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 NO: 71. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence that is 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 NO: 71. In some embodiments, a nucleotide sequence has about 1-10, e.g., 1-5, or 6-10 nucleotide substitutions compared to a sequence of SEQ ID NO: 71. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of the nucleotide sequence of SEQ ID NO: 71. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of the nucleotide sequence of SEQ ID NO: 71. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 71.

[0089] In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 72. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 72. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of a nucleotide sequence that is 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 NO: 72. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence that is 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 NO: 72. In some embodiments, a nucleotide sequence has about 1-20, e.g., 1-5, 6-10, 11-15, or 16-20 nucleotide substitutions compared to a sequence of SEQ ID NO: 72. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of the nucleotide sequence of SEQ ID NO: 72. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of the nucleotide sequence of SEQ ID NO: 72. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 72.

[0090] In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 82. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 82. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of a nucleotide sequence that is 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 NO: 82. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence that is 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 NO: 82. In some embodiments, a nucleotide sequence has about 1-10, e.g., 1-5, or 6-10 nucleotide substitutions compared to a sequence of SEQ ID NO: 82. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of the nucleotide sequence of SEQ ID NO: 82. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of the nucleotide sequence of SEQ ID NO: 82. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 82.

[0091] In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence of SEQ ID NO: 83. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is 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 NO: 83. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of a nucleotide sequence that is 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 NO: 83. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of a nucleotide sequence that is 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 NO: 83. In some embodiments, a nucleotide sequence has about 1-10, e.g., 1-5, 6-10, 11-15, or 16-20 nucleotide substitutions compared to a sequence of SEQ ID NO: 83. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists essentially of the nucleotide sequence of SEQ ID NO: 83. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA consists of the nucleotide sequence of SEQ ID NO: 83. In some embodiments, a nucleic acid comprising a nucleotide sequence that encodes a siRNA comprises a nucleotide sequence that is the complement of SEQ ID NO: 83.

[0092] In some aspects, a nucleic acid comprising a nucleotide sequence that encodes a siRNA, e.g., a siRNA comprising a nucleotide sequence of SEQ ID NO 10 or SEQ ID NO: 16 can be present in other suitable introns known in the art.

[0093] Variant bovine AAV capsid proteins

[0094] The present disclosure provides variant BAAV capsid proteins that increase tropism and / or transduction efficiency for macrophages. The variant BAAV capsids described herein show enhanced tropism for and transduction of macrophages. In some embodiments, the macrophages are mouse macrophages. In some embodiments, the macrophages are human macrophages.

[0095] Screening of a BAAV capsid library in mouse macrophages led to the identification of variant BAAV capsid proteins that are capable of replicating in macrophages. In some embodiments, a variant BAAV capsid protein of the disclosure possesses enhanced efficiency to transduce macrophages compared to the transduction efficiency of wild-type BAAV capsid proteins. In some embodiments, a variant BAAV capsid protein of the disclosure possesses enhanced tropism for macrophages compared to the transduction efficiency of wild-type BAAV capsid proteins.

[0096] SEQ ID NO: 25 corresponds to an example of a wild-type bovine AAV capsid sequence.

[0097] Wild-type BAAV capsid amino acid sequence:

[0098] MSFVDHPPDWLESIGDGFREFLGLEAGPPKPKANQQKQDNARGLVLPGYKYLGPGN GLDKGDPVNFADEVAREHDLSYQKQLEAGDNPYLKYNHADAEFQEKLASDTSFGG NLGKAVFQAKKRILEPLGLVETPDKTAPAAKKRPLEQSPQEPDSSSGVGKKGKQPAR KRLNFDDEPGAGDGPPPEGPSSGAMSTETEMRAAAGGNGGDAGQGAEGVGNASGD WHCDSTWSESHVTTTSTRTWVLPTYNNHLYLRLGSSNASDTFNGFSTPWGYFDFNR FHCHFSPRDWQRLINNHWGLRPKSMQVRIFNIQVKEVTTSNGETTVSNNLTSTVQIF ADSTYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGLVTGGSSQNQTDRNAFYCLE YFPSQMLRTGNNFEMVYKFENVPFHSMYAHSQSLDRLMNPLLDQYLWELQSTTSG GTLNQGNSATNFAKLTKTNFSGYRKNWLPGPMMKQQRFSKTASQNYKIPQGRNNS LLHYETRTTLDGRWSNFAPGTAMATAANDATDFSQAQLIFAGPNITGNTTTDANNL MFTSEDELRATNPRDTDLFGHLATNQQNATTVPTVDDVDGVGVYPGMVWQDRDIY YQGPIWAKIPHTDGHFHPSPLIGGFGLKSPPPQIFIKNTPVPANPATTFSPARINSFITQY STGQVAVKIEWEIQKERSKRWNPEVQFTSNYGAQDSLLWAPDNAGAYKEPRAIGSR

[0099] YLTNHL (SEQ ID NO: 25)

[0100] In some embodiments, the variant BAAV capsid proteins of the disclosure comprise random 5 amino acid insertions in the wild-type capsid amino acid sequence (e.g., as set forth in SEQ ID NO: 25). In some embodiments, the 5 amino acid insertion is VAAAL (SEQ ID NO: 34). In some embodiments, the 5 amino acid insertion is VAAAS (SEQ ID NO: 35). In some embodiments, the insertion is at position 45 of SEQ ID NO: 25. In some embodiments, the 5 amino acid insertion is GAAAT (SEQ ID NO: 37). In some embodiments, the insertion is at position 15 of SEQ ID NO: 25. The sequence of a variant capsid protein with a VAAAL (SEQ ID NO: 34) insertion at position 45 is shown below.

[0101] Variant BAAV capsid amino acid sequence with VAAAL (SEQ ID NO: 34) insertion at position 45:

[0102] MSFVDHPPDWLESIGDGFREFLGLEAGPPKPKANQQKQDNARGLVAAALVLPGYKY LGPGNGLDKGDPVNFADEVAREHDLSYQKQLEAGDNPYLKYNHADAEFQEKLASD TSFGGNLGKAVFQAKKRILEPLGLVETPDKTAPAAKKRPLEQSPQEPDSSSGVGKKG KQPARKRLNFDDEPGAGDGPPPEGPSSGAMSTETEMRAAAGGNGGDAGQGAEGVG NASGDWHCDSTWSESHVTTTSTRTWVLPTYNNHLYLRLGSSNASDTFNGFSTPWGY FDFNRFHCHFSPRDWQRLINNHWGLRPKSMQVRIFNIQVKEVTTSNGETTVSNNLTS TVQIFADSTYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGLVTGGSSQNQTDRNA FYCLEYFPSQMLRTGNNFEMVYKFENVPFHSMYAHSQSLDRLMNPLLDQYLWELQS TTSGGTLNQGNSATNFAKLTKTNFSGYRKNWLPGPMMKQQRFSKTASQNYKIPQGR NNSLLHYETRTTLDGRWSNFAPGTAMATAANDATDFSQAQLIFAGPNITGNTTTDA NNLMFTSEDELRATNPRDTDLFGHLATNQQNATTVPTVDDVDGVGVYPGMVWQD RDIYYQGPIWAKIPHTDGHFHPSPLIGGFGLKSPPPQIFIKNTPVPANPATTFSPARINSF ITQYSTGQVAVKIEWEIQKERSKRWNPEVQFTSNYGAQDSLLWAPDNAGAYKEPRA IGSRYLTNHL (SEQ ID NO: 26)

[0103] Contemplated herein are also variant rAAV capsid proteins of serotypes other than BAAV. In some embodiments, any one of the amino acid insertions described herein are in a corresponding region of the capsid protein of a serotype other than BAAV that is homologous to capsid protein of BAAV. In some embodiments, a variant rAAV capsid protein of a serotype other than BAAV is of any serotype other than BAAV (e.g., AAV1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13).

[0104] Provided herein are also nucleic acids that encode any one of the variant rBAAV capsid proteins disclosed herein. In some embodiments, a nucleic acid encoding a variant rBAAV capsid protein is comprised in a plasmid.

[0105] Provided herein are also variant bovine AAV particles. In some embodiments, a particle is an empty particle (e.g., one that does not contain a nucleic acid vector comprising a gene of interest). In some embodiments, an bovine AAV particle contains an AAV vector comprising a gene of interest. As used herein, “a gene of interest” is a gene that encodes a RNA or protein of interest. In some embodiments, the AAV vector comprises a nucleotide sequence encoding an RNAi molecule that targets the PiZ allele of AAT. In some embodiments, the AAV vector further comprises a nucleotide sequence encoding PiM-AAT. The AAV vector can comprise ITRs from BAAV or from another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, etc.). The AAV vectors can also comprise additional regulatory elements as described herein elsewhere. Method, of making rAAV particles

[0106] Various methods of producing rAAV particles and nucleic acid vectors are known (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). In some embodiments, a vector (e.g., a plasmid) comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP region as described herein), and transfected into a recombinant cells, called helper or producer cells, such that the nucleic acid vector is packaged or encapsidated inside the capsid and subsequently purified.

[0107] Non-limiting examples of mammalian helper cells include HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, e.g., ATCC® CRL-1573™, ATCC® CRL- 1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-10™, or ATCC® CCL- 61™). A non-limiting example of an insect helper cells is Sf9 cells (see, e.g., ATCC® CRL- 1711™). A helper cell may comprises rep and / or cap genes that encode the Rep protein and / or Cap proteins. In some embodiments, the packaging is performed in vitro (e.g., outside of a cell).

[0108] In some embodiments, a nucleic acid vector (e.g., a plasmid) containing the gene of interest is combined with one or more helper plasmids, e.g., that contain a rep gene of a first serotype and a cap gene of the same serotype or a different serotype, and transfected into helper cells such that the rAAV particle is packaged. In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene, and a second helper plasmid comprising one or more of the following helper genes: Ela gene, Elb gene, E4 gene, E2a gene, and VA gene. For clarity, helper genes are genes that encode helper proteins Ela, Elb, E4, E2a, and VA. Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDF6, pRep, pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno- Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno- Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). Plasmids that encode wild-type AAV coding regions for specific serotypes are also know and available. For example pSub201 is a plasmid that comprises the coding regions of the wild-type AAV2 genome (Samulski et al. (1987), J Virology, 6:3096-3101).

[0109] ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24): 14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385 / 1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Pat. Nos. 5,139,941 and 5,962,313, all of which are incorporated herein by reference).

[0110] Genbank reference numbers for sequences of AAV serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 are listed in patent publication W02012064960, which is incorporated herein by reference in its entirety.

[0111] A non-limiting method of rAAV particle production method is described next. One or more helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids comprise rep genes, cap genes, and optionally one or more of the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids comprise cap ORFs (and optionally rep ORFs) for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The cap ORF may also comprise one or more modifications to produce a modified capsid protein as described herein. As an example, HEK293 cells (available from ATCC®) are transfected via CaPO4-mediated transfection, lipids or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector. The HEK293 cells are then incubated for at least 60 hours to allow for rAAV particle production. Alternatively, the HEK293 cells are transfected via methods described above with AAV-ITR containing one or more genes of interest, a helper plasmid comprising genes encoding Rep and Cap proteins, and co-infected with a helper virus. Helper viruses are viruses that allow the replication of AAV. Examples of helper virus are adenovirus and herpesvirus.

[0112] Alternatively, in another example, Sf9-based producer stable cell lines are infected with a single recombinant baculovirus containing the nucleic acid vector. As a further alternative, in another example HEK293 or BHK cell lines are infected with a HSV containing the nucleic acid vector and optionally one or more helper HSVs containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow for rAAV particle production. The rAAV particles can then be purified using any method known in the art or described herein, e.g., by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0113] Methods for large-scale production of AAV using a herpesvirus-based system are also known. See for example, Clement et al. (Hum Gene Ther. 2009, 20(8):796-806). Methods of producing exosome-associated AAV, which can be more resistant to neutralizing anti-AAV antibodies, are also known (Hudry et al., Gene Ther. 2016, 23(4):380-92; Macguire et al., Mol Ther. 2012, 20(5):960-71).

[0114] Methods for producing and using pseudotyped rAAV vectors are also known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524- 1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

[0115] Compositions

[0116] Various formulations have been developed to facilitate rAAV particle use. For example, for administration of an injectable aqueous solution of rAAV particles, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. In some embodiments, a composition as provided herein comprises a plurality of any one of the rAAV particles disclosed herein. In some embodiments, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful.

[0117] Accordingly, in some embodiments, a composition of variant rAAV particles comprises a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particle is administered. Such pharmaceutical carriers can be sterile liquids (e.g., water, oils,, saline solutions, aqueous dextrose and glycerol solutions), suspending agents, preserving agents (e.g., methyl-, ethyl-, and propyl-hydroxy-benzoates), and pH adjusting agents (such as inorganic and organic acids and bases). In some embodiments, carriers include buffered saline solutions (e.g., phosphate buffered saline, HEPES -buffered saline). USP grade carriers and excipients are particularly useful for delivery of rAAV particles to human subjects. Such compositions may further optionally comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. Methods for making such compositions 1 are well known and can be found in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2012.

[0118] In some embodiments, a composition comprising any one of the rAAV particles disclosed herein comprises Balanced Salt Solution (BSS) supplemented with 0.014% Tween 20 (polysorbate 20).

[0119] Typically, compositions may contain at least about 0.1% of the therapeutic agent (e.g., rAAV particle) or more, although the percentage of the active ingredient(s) may 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 therapeutic agent(s) (e.g., rAAV particle) in each therapeutically-useful composition may be prepared is 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.

[0120] The pharmaceutical forms of rAAV particle compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. In some embodiments, the form is sterile. The carrier can be a solvent or dispersion medium containing, for example, water, saline, 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.

[0121] Preparation of compositions for administration to a subject are known in the art. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by, e.g., FDA Office of Biologies standards.

[0122] Methods of Use

[0123] Any one of the rAAV particles, or compositions comprising any one of the rAAV particles disclosed herein can be used to transduce a cell, tissue or organ.

[0124] In some embodiments, a cell, tissue or organ is transduced in an in vitro setting wherein the cell, tissue or organ is incubated or perfused with a media. A cell may be one of many cells cultured under certain conditions, or part of an organ that is harvested, part of an organoid, or an organism.

[0125] In some embodiments, a cell, tissue or organ is transduced in vivo, for example, for the purposes of treating A1AD. In some embodiments, provided herein is a method of transducing a cell or tissue of the lung or liver. In some embodiments, a specific tissue in the lung or liver is targeted. In some embodiments, a specific cell type in the lung or liver is targeted. For example, in some embodiments, macrophages in the lungs are targeted. In some embodiments, hepatocytes in the liver are targeted.

[0126] In some embodiments, a subject in which a cell, tissue or organ is transduced is a vertebrate animal (e.g., a mammal or reptile). In some embodiments, a mammalian subject is a human, a non-human primate, a dog, a cat, a hamster, a mouse, a rat, a pig, a horse, a cow, a donkey or a rabbit. Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus or rhesus macaques), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. In some embodiments, a subject is a model for a particular disease or used to study the pharmacokinetics and / or pharmacokinetics of a protein or siRNA encoded by encoded by the recombinant nucleic acid in an rAAV of the disclosure.

[0127] In some aspects, the present disclosure provides methods of treating A1AD. In one aspect, the disclosure provides a method of treating A1AD in a subject in need thereof, comprising administering to the subject an rAAV vector or rAAV particle of the disclosure.

[0128] To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered. For example, an effective amount of rAAV particles may be an amount of the particles that are capable of transferring an expression construct to a host cell, tissue or organ. A therapeutically acceptable amount may be an amount that is capable of treating a disease, e.g., A1AD. As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

[0129] The terms “subject,” and “patient,” are used interchangeably herein. In some embodiments, a subject is a mammal, such as a human, a nonhuman primate, a dog, a cat, a horse, a sheep, a poultry, a cow, a pig, a mouse, a rat, a rodent, or a goat. In some embodiments, the subject and mammal is a human.

[0130] Methods of Administration

[0131] Any of the nucleic acids, AAV vectors, AAV particles, or compositions disclosed herein may be administered to a subject.

[0132] Suitable routes of administration include, without limitation, intravenous, intranasal, intramuscular, intrathecal, or subcutaneous. In some embodiments, a polypeptide, engineered nucleic acid, vector, or composition of the disclosure is administered intravenously, subcutaneously, intramuscularly intrathecally or intranasally. In some embodiments, a nucleic acid, vector, or composition of the disclosure is administered directly (e.g., by direct injection) to one or more cells (e.g., liver or lung cells), tissues, or organs (e.g., liver or lung). Other routes of administration are contemplated herein. The administration route can be changed depending on a number of factors, including the desired cell, tissue, or organ.

[0133] Formulations comprising pharmaceutically-acceptable excipients and / or carrier solutions are 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, including e.g., oral, parenteral, intravenous, intranasal, intraarticular, and intramuscular administration and formulation.

[0134] EXAMPLES

[0135] Example 1: Multifunctional sc AAV vectors

[0136] The rationale behind using gene silencing to target the PiZ mutation is to prevent the accumulation of the PiZ polymers in the cells, especially in the liver. Less PiZ polymers in the liver will reduce the cellular stress and allow tissue repair and possible reverse of liver fibrosis associated with repeat cellular damage. In macrophages, lowering PiZ accumulation will help to restore normal cellular function and protect the lung from excessive inflammatory response.

[0137] Multifunctional scAAV vectors that comprise (i) a first RNA molecule: a PiZ-specific siRNA to silence the PiZ allele; and (ii) a second RNA molecule: the PiM mRNA to upregulate expression of healthy AAT, were developed. scAAV allows for higher in vivo DNA stability and more effective circulation of the vector genome. Rapid and long-term RNA transcription results in higher gene expression is another advantage of scAAV when compared to single-strand AAV vectors. One challenge of using scAAV vectors is the limited packaging capacity. To overcome this barrier, several short gene expression cassettes have been developed that fit within the limited packaging capacity. An exemplary vector comprising a packaging insert of 2.7 kb is shown in the right panel of FIG. 2.

[0138] To deliver the PiZ-specific RNAi molecule without affecting the PiM mRNA molecule in the scAAV vector, the intron in the vector was repurposed to encode the PiZ- specific siRNA, plO. See FIGS. 1 and 2. By using the intron to encode the RNAi, PiM mRNA undergoes normal processing and elicits long-term protein expression. Achieving long term PiM expression in both the liver and the lung alleviates A1AD symptoms and restores protective levels of AAT in the serum.

[0139] The PiZ-specific RNAi molecule was inserted into the intron of the CAG promoter or into the SV40 intron. The schematic for CAG promoter modification is shown in FIG. 3A. The schematic for SV40 intron modification is shown in FIG. 3B. Exemplary AAV vectors comprising plO siRNA inserted into the CAG intron or the SV40 intron are shown in FIGS. 4A and 4B, respectively.

[0140] Example 2: PiZ-specific silencing

[0141] Allele specific siRNA that can preferentially target the PiZ mutant AAT over the normal PiM AAT were created. The initial design of the siRNA was done by creating every possible siRNA around the single base pair change differentiating PiZ and PIM AAT (FIG. 5A). It has been shown previously that mismatches at position 10 (p 10) and position 16 (p 16) have the highest affinity for allelic differentiation, thus shRNAs were designed based on these positions for further studies. The sequence of p 10 as a hairpin RNA is shown in FIG. 5B. An AAV plasmid encoding the PiZ-specific siRNA plO is shown in FIG. 6.

[0142] In vitro studies were carried out on cells secreting either wildtype or mutant AAT with allele specific shRNA constructs. Cells that expressed either PiZ (mutant) or PiM (wildtype) AAT were transfected with plasmids expressing either the plO or pl6 shRNA. AAT levels in the supernatants were assessed by ELISA at 48 and 72 hrs. The data is expressed as percent knockdown of AAT as compared to cells transfected with a negative control (NC) shRNA (FIG. 7). The plO siRNA had a higher specificity ratio for the PiZ allele over PiM.

[0143] The in vivo effects of the plO shRNA were also studied. FIG. 8 shows the results of IHC staining for human AAT from the livers of human PiZ transgenic mice after portal vein delivery of rAAV8-U6-plO shRNA or negative control (NC) vector. Total serum AAT levels were measured in PiZ mice after portal vein injections with allele specific plO shRNA or negative control packaged in rAAV8. Mice transgenic for the human PiZ gene were used to assess the efficacy of rAAV-shRNA mediated knockdown of AAT in vivo. Two sets of experiments are shown in FIG. 9, a 2- week experiment and a 4- week experiment. Thus, plO is effective in silencing the PiZ allele.

[0144] AAV plasmids that allow simultaneous Piz- specific silencing and M-AAT augmentation were designed. An exemplary plasmid is shown in FIG. 10.

[0145] Free energy of plO precursor and processed RNAi molecules is shown in FIG. 11. AAT levels were measured in cell supernatants (FIG. 12) and cell pellets (FIG. 13) in the presence of miRNAplO (precursor), plO siRNA, a positive control (3X), and a negative control (UF11).

[0146] Example 3: Development of AAV capsids for macrophage targeting

[0147] Bovine AAV has higher tropism for mouse and human macrophages when compared to other AAV viruses (FIGs. 14A-14D). Taking advantage of the higher tropism of BAAV for mouse and human macrophages when compared to other AAV viruses. To increase the tropism of BAAV for macrophages, a capsid library of BAAV mutants was developed by using the transposition machinery of the bacteriophage Mu (ThermoFisher kit). Mu transposon generates a random five-peptide-long insertion in the capsid (FIG. 15). Since several viruses can infect and replicate in macrophages cells, a similar approach was used. Primary adult mouse macrophages derived from the spleen were infected for several days (biopanning technique) with the BAAV library to isolate possible new BAAV variants capable of replicating. Only a small number of variants were sequenced (10) but the VAAA- X (SEQ ID NO: 32) (X - can be L or S) at position 45 of the capsid showed up 3 times and variant GAAA-X (SEQ ID NO: 33) (X - can be A or T) at position 15 of the capsid showed up twice, suggesting that the identified insertions enhance tropism for macrophages. Table 1 below lists the naive and macrophage-rescued capsids.

[0148] Table 1. Capsid library results

[0149] Example 4: Development of a truncated human CD68 promoter

[0150] A truncated human CD68 promoter was developed and studied. The truncated promoter is 347 bp in length and its sequence is shown in FIG. 21. An exemplary AAV vector comprising the truncated hCD68 promoter operably linked to a gene encoding a red fluorescent protein (RFP) is shown in FIG. 22.

[0151] The ability of the truncated promoter to express genes in macrophages was demonstrated by determining mCherry expression in microglia (brain macrophages). Colocalization of GFP and RFP in CX3CR1-GFP mice brain slices transfected with dsAAV- CD68-MVMi-mCherry demonstrated localization of the truncated hCD68 promoter in microglia (brain macrophages) (FIG. 23).

[0152] A dsAAV-CD68-MVMi-hGFP vector was also developed. Brain and spinal cord slice cultures from mice transfected with dsAAV-CD68-MVMi-hGFP are shown in FIG. 24. GFP expression was detected in brain slice and spinal cord slice cultures.

[0153] Sequences dsAAV-minCMV-SV40-P10-intron-MAAT-kpnl-Agel-strategy-pLann sequence (SEQ ID NO: 21): ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggaattcacgcgtggatctgaattcaattcacgcgtggtaccaaaatcaacgggactttccaaaatgtc gtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtca gaattctcgagtgatcgaaagagcctgctaaagcaaaaaagaagtcaccgtcgacgaggaactgaaaaaccagaaagttaactggta agtttagtctttttgtcttttatttcaggtcccgctagcaccatcgacaagaaagggattcaagagatccctttcttgtcgatggtgaattcgg atccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaag ctgcggaattgtacccgcggccgatccaccggtacgcgttctagaaagcttgatatccctgcagggcggcccgaattcgcccttatgc cgtcttctgtctcgtggggcatcctcctgctggcaggcctgtgctgcctggtccctgtctccctggctgaggatccccagggagatgctg cccagaagacagatacatcccaccatgatcaggatcacccaaccttcaacaagatcacccccaacctggctgagttcgccttcagcct ataccgccagctggcacaccagtccaacagcaccaatatcttcttctccccagtgagcatcgctacagcctttgcaatgctctccctggg gaccaaggctgacactcacgatgaaatcctggagggcctgaatttcaacctcacggagattccggaggctcagatccatgaaggcttc caggaactcctccgtaccctcaaccagccagacagccagctccagctgaccaccggcaatggcctgttcctcagcgagggcctgaa gctagtggataagtttttggaggatgttaaaaagttgtaccactcagaagccttcactgtcaacttcggggacaccgaagaggccaaga aacagatcaacgattacgtggagaagggtactcaagggaaaattgtggatttggtcaaggagcttgacagagacacagtttttgctctg gtgaattacatcttctttaaaggcaaatgggagagaccctttgaagtcaaggacaccgaggaagaggacttccacgtggaccaggtga ccaccgtgaaggtgcctatgatgaagcgtttaggcatgtttaacatccagcactgtaagaagctgtccagctgggtgctgctgatgaaat acctgggcaatgccaccgccatcttcttcctgcctgatgaggggaaactacagcacctggaaaatgaactcacccacgatatcatcac caagttcctggaaaatgaagacagaaggtctgccagcttacatttacccaaactgtccattactggaacctatgatctgaagagcgtcct gggtcaactgggcatcactaaggtcttcagcaatggggctgacctctccggggtcacagaggaggcacccctgaagctctccaagg ccgtgcataaggctgtgctgaccatcgacgagaaagggactgaagctgctggggccatgtttttagaggccatacccatgtctatcccc cccgaggtcaagttcaacaaaccctttgtcttcttaatgattgaacaaaataccaagtctcccctcttcatgggaaaagtggtgaatccca cccaaaaataacaagggcgaattcgtttaaacctgcaggactagcgagctcgctgatcagcctcgactgtgccttctagttgccagcca tctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcat tgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgct ggggagagattgatctgaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgacc aaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggcccccccccccc cccccccggcgattctcttgtttgctccagactctcaggcaatgacctgatagcctttgtagagacctctcaaaaatagctaccctctccg gcatgaatttatcagctagaacggttgaatatcatattgatggtgatttgactgtctccggcctttctcacccgtttgaatctttacctacacat tactcaggcattgcatttaaaatatatgagggttctaaaaatttttatccttgcgttgaaataaaggcttctcccgcaaaagtattacagggtc ataatgtttttggtacaaccgatttagctttatgctctgaggctttattgcttaattttgctaattctttgccttgcctgtatgatttattggatgttg gaatcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgc atagttaagccagccccgacacccgccaacactatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccga cacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagc tgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatga taataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat ccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcc cttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgg gttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctg ctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtac tcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcgg ccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgtt gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctc ggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatg gtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgc ctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggt gaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagga tcttcttgagatccttttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaaga gctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat ccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtt tcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttac ggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagct gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcc tctccccgcgcgttggccgattcattaatgcagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagc ctgaatggcgaatggcgattccgttgcaatggctggcggtaatattgttctggatattaccagcaaggccgatagtttgagttcttctactc aggcaagtgatgttattactaatcaaagaagtattgcgacaacggttaatttgcgtgatggacagactcttttactcggtggcctcactgat tataaaaacacttctcaggattctggcgtaccgttcctgtctaaaatccctttaatcggcctcctgtttagctcccgctctgattctaacgag gaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcg cagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtc aagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacg tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaa cactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaattta acgcgaattttaacaaaatattaacgcttacaatttaaatatttgcttatacaatcttcctgtttttggggcttttctgattatcaaccggggtac atatgattgacatgctagttttacgattaccgttcatcgcc dsAAV-hCD68-SV40-pl0-intron-MAAT-KpnI-AgeI-strategy_pLann sequence (SEQ ID NO: 22): ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggaattcacgcgtggatctgaattcaattcacgcgtggtaccttaattacaaaaactaatgactaagag agaggtggctagagctgaggcccctgagtcaggctgtgggtgggatcatctccagtacaggaagtgagactttcatttcctcctttcca agagagggctgagggagcagggttgagcaactggtgcagacagcctagctggactttgggtgaggcggttcagccatatcgaattct gctggggctactggcaggtaaggaggaaggaggctgaggggagggggcccctgggagggagcctgccctgggttgctaaccatc tcctctctgccaaaagcccaggggacactagtgaagcttaaagtcgacgaggaactgaaaaaccagaaagttaactggtaagtttagt ctttttgtcttttatttcaggtcccgctagcaccatcgacaagaaagggattcaagagatccctttcttgtcgatggtgaattcggatccggt ggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcgg aattgtacccgcggccgatccaccggtacgcgttctagaaagcttgatatccctgcagggcggcccgaattcgcccttatgccgtcttct gtctcgtggggcatcctcctgctggcaggcctgtgctgcctggtccctgtctccctggctgaggatccccagggagatgctgcccaga agacagatacatcccaccatgatcaggatcacccaaccttcaacaagatcacccccaacctggctgagttcgccttcagcctataccgc cagctggcacaccagtccaacagcaccaatatcttcttctccccagtgagcatcgctacagcctttgcaatgctctccctggggaccaa ggctgacactcacgatgaaatcctggagggcctgaatttcaacctcacggagattccggaggctcagatccatgaaggcttccaggaa ctcctccgtaccctcaaccagccagacagccagctccagctgaccaccggcaatggcctgttcctcagcgagggcctgaagctagtg gataagtttttggaggatgttaaaaagttgtaccactcagaagccttcactgtcaacttcggggacaccgaagaggccaagaaacagat caacgattacgtggagaagggtactcaagggaaaattgtggatttggtcaaggagcttgacagagacacagtttttgctctggtgaatta catcttctttaaaggcaaatgggagagaccctttgaagtcaaggacaccgaggaagaggacttccacgtggaccaggtgaccaccgt gaaggtgcctatgatgaagcgtttaggcatgtttaacatccagcactgtaagaagctgtccagctgggtgctgctgatgaaatacctgg gcaatgccaccgccatcttcttcctgcctgatgaggggaaactacagcacctggaaaatgaactcacccacgatatcatcaccaagttc ctggaaaatgaagacagaaggtctgccagcttacatttacccaaactgtccattactggaacctatgatctgaagagcgtcctgggtcaa ctgggcatcactaaggtcttcagcaatggggctgacctctccggggtcacagaggaggcacccctgaagctctccaaggccgtgcat aaggctgtgctgaccatcgacgagaaagggactgaagctgctggggccatgtttttagaggccatacccatgtctatcccccccgagg tcaagttcaacaaaccctttgtcttcttaatgattgaacaaaataccaagtctcccctcttcatgggaaaagtggtgaatcccacccaaaaa taacaagggcgaattcgtttaaacctgcaggactagcgagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgttt gcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgag taggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggag agattgatctgaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtc gcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccccccccccccccccccc ggcgattctcttgtttgctccagactctcaggcaatgacctgatagcctttgtagagacctctcaaaaatagctaccctctccggcatgaat ttatcagctagaacggttgaatatcatattgatggtgatttgactgtctccggcctttctcacccgtttgaatctttacctacacattactcagg cattgcatttaaaatatatgagggttctaaaaatttttatccttgcgttgaaataaaggcttctcccgcaaaagtattacagggtcataatgttt ttggtacaaccgatttagctttatgctctgaggctttattgcttaattttgctaattctttgccttgcctgtatgatttattggatgttggaatcgcc tgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaa gccagccccgacacccgccaacactatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgc caacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgt gtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataat ggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctc atgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttg cggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacat cgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtg gcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcacca gtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactt acttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaac cggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactg gcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggccctt ccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagc cctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcact gattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttg agatccttttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctacca actctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaaga actctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttgga ctcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacc tacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaa gcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccac ctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctg gccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgc tcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccg cgcgttggccgattcattaatgcagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatggcgattccgttgcaatggctggcggtaatattgttctggatattaccagcaaggccgatagtttgagttcttctactcaggcaa gtgatgttattactaatcaaagaagtattgcgacaacggttaatttgcgtgatggacagactcttttactcggtggcctcactgattataaaa acacttctcaggattctggcgtaccgttcctgtctaaaatccctttaatcggcctcctgtttagctcccgctctgattctaacgaggaaagca cgttatacgtgctcgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtg accgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctct aaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgg gccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactca accctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaatattaacgcttacaatttaaatatttgcttatacaatcttcctgtttttggggcttttctgattatcaaccggggtacatatgat tgacatgctagttttacgattaccgttcatcgcc dsAAV-CD68-MVMi-hGFP-Genscript_pLann sequence (SEQ ID NO: 23): ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggaattcacgcgtggatctgaattcaattcacgcgtggtaccttaattacaaaaactaatgactaagag agaggtggctagagctgaggcccctgagtcaggctgtgggtgggatcatctccagtacaggaagtgagactttcatttcctcctttcca agagagggctgagggagcagggttgagcaactggtgcagacagcctagctggactttgggtgaggcggttcagccatatcgaattct gctggggctactggcaggtaaggaggaaggaggctgaggggagggggcccctgggagggagcctgccctgggttgctaaccatc tcctctctgccaaaagcccaggggacactagtgaagcttaaagtcgacaagaggtaagggtttaagggatggttggttggtggggtatt aatgtttaattacctgttttacaggcctgaaatcacttggttttaggttgggaagcttgatatccctgcagggcggccgccgccaccatga gcaagggcgaggaactgttcactggcgtggtcccaattctcgtggaactggatggcgatgtgaatgggcacaaattttctgtcagcgg agagggtgaaggtgatgccacatacggaaagctcaccctgaaattcatctgcaccactggaaagctccctgtgccatggccaacact ggtcactaccctgacctatggcgtgcagtgcttttccagatacccagaccatatgaagcagcatgactttttcaagagcgccatgcccga gggctatgtgcaggagagaaccatctttttcaaagatgacgggaactacaagacccgcgctgaagtcaagttcgaaggtgacaccct ggtgaatagaatcgagctgaagggcattgactttaaggaggatggaaacattctcggccacaagctggaatacaactataactcccac aatgtgtacatcatggccgacaagcaaaagaatggcatcaaggtcaacttcaagatcagacacaacattgaggatggatccgtgcagc tggccgaccattatcaacagaacactccaatcggcgacggccctgtgctcctcccagacaaccattacctgtccacccagtctgccctg tctaaagatcccaacgaaaagagagaccacatggtcctgctggagtttgtgaccgctgctgggatcacacatggcatggacgagctgt acaagtgagcggccgcgctagcgagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggagagatcgatctgag gaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgccc gggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggcccccccccccccccccccggcgattctcttg tttgctccagactctcaggcaatgacctgatagcctttgtagagacctctcaaaaatagctaccctctccggcatgaatttatcagctagaa cggttgaatatcatattgatggtgatttgactgtctccggcctttctcacccgtttgaatctttacctacacattactcaggcattgcatttaaa atatatgagggttctaaaaatttttatccttgcgttgaaataaaggcttctcccgcaaaagtattacagggtcataatgtttttggtacaaccg atttagctttatgctctgaggctttattgcttaattttgctaattctttgccttgcctgtatgatttattggatgttggaatcgcctgatgcggtattt tctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccga cacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagc tgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatga taataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat ccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcc cttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgg gttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctg ctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtac tcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcgg ccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgtt gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctc ggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatg gtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgc ctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggt gaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagga tcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagag ctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccact tcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgg gttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcga acgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatc cggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggttt cgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacg gttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctg ataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcct ctccccgcgcgttggccgattcattaatgcagctgattctaacgaggaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcg ccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcct ttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgtt ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgc cgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaaatatttgct tatacaatcttcctgtttttggggcttttctgattatcaaccggggtacatatgattgacatgctagttttacgattaccgttcatcgcc dsAAV-minCMV-SV40i-pl0-RNAi-hGFP_pLann sequence (SEQ ID NO: 24): ggtaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggagg tctatataagcagagctcgtttagtgaaccgtcagaattctcgagtgatcgaaagagcctgctaaagcaaaaaagaagtcaccgtcgac gaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttatttcaggtcccgctagcaccatcgacaagaaagggatt caagagatccctttcttgtcgatggtgaattcggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttcta ggcctgtacggaagtgttacttctgctctaaaagctgcggaattgtacccgcggccgatccaccggtacgcgttctagaaagcttgatat ccctgcagggcggccgccgccaccatgagcaagggcgaggaactgttcactggcgtggtcccaattctcgtggaactggatggcga tgtgaatgggcacaaattttctgtcagcggagagggtgaaggtgatgccacatacggaaagctcaccctgaaattcatctgcaccactg gaaagctccctgtgccatggccaacactggtcactaccctgacctatggcgtgcagtgcttttccagatacccagaccatatgaagcag catgactttttcaagagcgccatgcccgagggctatgtgcaggagagaaccatctttttcaaagatgacgggaactacaagacccgcg ctgaagtcaagttcgaaggtgacaccctggtgaatagaatcgagctgaagggcattgactttaaggaggatggaaacattctcggcca caagctggaatacaactataactcccacaatgtgtacatcatggccgacaagcaaaagaatggcatcaaggtcaacttcaagatcaga cacaacattgaggatggatccgtgcagctggccgaccattatcaacagaacactccaatcggcgacggccctgtgctcctcccagac aaccattacctgtccacccagtctgccctgtctaaagatcccaacgaaaagagagaccacatggtcctgctggagtttgtgaccgctgc tgggatcacacatggcatggacgagctgtacaagtgagcggccgcgctagcgagctcgctgatcagcctcgactgtgccttctagttg ccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattg catcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagca ggcatgctggggagagatcgatctgaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggcc gggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccc ccccccccccccccccggcgattctcttgtttgctccagactctcaggcaatgacctgatagcctttgtagagacctctcaaaaatagcta ccctctccggcatgaatttatcagctagaacggttgaatatcatattgatggtgatttgactgtctccggcctttctcacccgtttgaatcttta cctacacattactcaggcattgcatttaaaatatatgagggttctaaaaatttttatccttgcgttgaaataaaggcttctcccgcaaaagtat tacagggtcataatgtttttggtacaaccgatttagctttatgctctgaggctttattgcttaattttgctaattctttgccttgcctgtatgatttat tggatgttggaatcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctct gatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttac agacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtg atacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctattt gtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgag tattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatg ctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgt tttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatac actattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctg ccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatg ggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgta gcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcgga taaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcg gtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacg aaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaa acttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgt cagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacc agcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcctt ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggtt cgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttccc gaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacg cctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa aacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggata accgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcgga agagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcgtaatagcgaagaggcccgcaccg atcgcccttcccaacagttgcgcagcctgaatggcgaatggcgattccgttgcaatggctggcggtaatattgttctggatattaccagc aaggccgatagtttgagttcttctactcaggcaagtgatgttattactaatcaaagaagtattgcgacaacggttaatttgcgtgatggaca gactcttttactcggtggcctcactgattataaaaacacttctcaggattctggcgtaccgttcctgtctaaaatccctttaatcggcctcctg tttagctcccgctctgattctaacgaggaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcgccctgtagcggcgcattaa gcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttc tcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaa aaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata gtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggtta aaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaaatatttgcttatacaatcttcctgtttttg gggcttttctgattatcaaccggggtacatatgattgacatgctagttttacgattaccgttcatcgccctgcgcgctcgctcgctcactga ggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtgg aattcacgcgtggatctgaattcaattcacgcg

[0154] Bov-45-Genscript_pLann (SEQ ID NO: 28) atgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttgga actaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctg tttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaactggaccaat gagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagtcggcc aaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcac ctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaattt gaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtgg ttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaa cgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcggacaggtaccaaaacaaatgttct cgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagactgaatcagaattcaaatatctgcttcactcacggtgtca aagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcacatc atgggaaaggtgccagacgcttgcactgcttgcgacctggtcaatgtggacttggatgactgtgtttctgaacaataaatgacttaaacc aggtatgtcttttgttgaccaccctccagattggttggaatcgatcggcgacggctttcgtgaatttctcggccttgaggcgggtcccccg aaacccaaggccaatcaacagaagcaagataacgctcgaggtcttgttgcggccgcacttgtgcttcctgggtacaagtatcttggtcc tgggaacggccttgataagggcgatcctgtcaattttgctgacgaggttgcccgagagcacgacctctcctaccagaaacagcttgag gcgggcgataacccttacctcaagtacaaccacgcggacgcagagtttcaggagaaactcgcttctgacacttcttttgggggaaacct tgggaaggctgttttccaggctaaaaagaggattctcgaacctcttggcctggttgagacgccggataaaacggcgcctgcggcaaaa aagaggcctctagagcagagtcctcaagagccagactcctcgagcggagttggcaagaaaggcaaacagcctgccagaaagaga ctcaactttgacgacgaacctggagccggagacgggcctcccccagaaggaccatcttccggagctatgtctactgagactgaaatg cgtgcagcagctggcggaaatggtggcgatgcgggacaaggtgccgagggagtgggtaatgcctccggtgattggcattgcgattc cacttggtcagagagccacgtcaccaccacctcaacccgcacctgggtcctgccgacctacaacaaccacctgtacctgcggctcgg ctcgagcaacgccagcgacaccttcaacggattctccaccccctggggatactttgactttaaccgcttccactgccacttctcgccaag agactggcaaaggctcatcaacaaccactggggactgcgccccaaaagcatgcaagtccgcatcttcaacatccaagttaaggaggt cacgacgtctaacggggagacgaccgtatccaacaacctcaccagcacggtccagatctttgcggacagcacgtacgagctcccgt acgtgatggatgcaggtcaggagggcagcttgcctcctttccccaacgacgtgttcatggtgcctcagtacgggtactgcggactggt aaccggaggcagctctcaaaaccagacagacagaaatgccttctactgtctggagtactttcccagccagatgctgagaaccggaaa caactttgagatggtgtacaagtttgaaaacgtgcccttccactccatgtacgctcacagccagagcctggataggctgatgaacccgc tgctggaccagtacctgtgggagctccagtctaccacctctggaggaactctcaaccagggcaattcagccaccaactttgccaagct gaccaaaacaaacttttctggctaccgcaaaaactggctcccggggcccatgatgaagcagcagagattctccaagactgccagtca aaactacaagattccccagggaagaaacaacagtctgctccattatgagaccagaactaccctcgacggaagatggagcaattttgcc ccgggaacggccatggcaaccgcagccaacgacgccaccgacttctctcaggcccagctcatctttgcggggcccaacatcaccgg caacaccaccacagatgccaataacctgatgttcacttcagaagatgaacttagggccaccaacccccgggacactgacctgtttggc cacctggcaaccaaccagcaaaacgccaccaccgttcctaccgtagacgacgtggacggagtcggcgtgtacccgggaatggtgtg gcaggacagagacatttactaccaagggcccatttgggccaaaattccacacacggatggacactttcacccgtctcctctcattggcg gatttggactgaaaagcccgcctccacaaatattcatcaaaaacactcctgtacccgccaatcccgcaacgaccttctctccggccaga atcaacagcttcatcacccagtacagcaccggacaggtggctgtcaaaatagaatgggaaatccagaaggagcggtccaagagatg gaacccagaggtccagttcacgtccaactacggagcacaggactcgcttctctgggctcccgacaacgccggagcctacaaagagc ccagggccattggatcccgatacctcaccaaccacctctagcccaattctgttgcataccctcaataaaccgtacctccgacctgagga gatcgatgtgccctgcaggactagagtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggt atttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcca tcacactggcggccgctcgactagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttg gcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaag cctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgc attaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtc gttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacat gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagc atcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcg tgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgt aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccgg taactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtat gtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcc agttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagatt acgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaactt ggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgt agataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatc agcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgg gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtat ggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctcc gatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagat gctttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacggga taataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgtt gagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacagga aggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatc agggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcc acctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatc ccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaac gtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgta aagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaaggga agaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatg cgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcc agctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggcca gtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccccctcgatcgaggtcgacggtatcgggggagctcg cagggtctccattttgaagcgggaggtttgaacgcgcagccgccatgccggggttttacgagattgtgattaaggtccccagcgacctt gacgagcatctgcccggcatttctgacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatc tgaatctgattgagcaggcacccctgaccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccc cggaggctcttttctttgtgcaatttgagaagggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatg gttttgggacgtttcctgagtcagattcgcgaaaaactgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcg gtcacaaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattacttgctccccaaaaccca gcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcag catctgacgcacgtgtcgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaact tcagccaggtacatggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggatccaggaggaccaggcct catacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggtgccttggacaatgcgggaaagatt dsAAV-minCMV-intron-plO-MAAT_pLaan (SEQ ID NO: 59) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggaattcacgcgtggatctgaattcaattcacgcgtggtaccaaaatcaacgggactttccaaaatgtc gtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtca gaattctcgagtgatcgaaagagcctgctaaagcaaaaaagaagtcaccgtcgacgaggaactgaaaaaccagaaagttaactggta agtttagtctttttgtcttttatttcaggtcccgctagcaccatcgacaagaaagggattcaagagatccctttcttgtcgatggtgaattcgg atccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaag ctgcggaattgtacccgcggccgatccaccggtacgcgttctagaaagcttgatatccctgcagggcggccgcgaattcgccctttatg ccgtcttctgtctcgtggggcatcctcctgctggcaggcctgtgctgcctggtccctgtctccctggctgaggatccccagggagatgct gcccagaagacagatacatcccaccatgatcaggatcacccaaccttcaacaagatcacccccaacctggctgagttcgccttcagcc tataccgccagctggcacaccagtccaacagcaccaatatcttcttctccccagtgagcatcgctacagcctttgcaatgctctccctgg ggaccaaggctgacactcacgatgaaatcctggagggcctgaatttcaacctcacggagattccggaggctcagatccatgaaggctt ccaggaactcctccgtaccctcaaccagccagacagccagctccagctgaccaccggcaatggcctgttcctcagcgagggcctga agctagtggataagtttttggaggatgttaaaaagttgtaccactcagaagccttcactgtcaacttcggggacaccgaagaggccaag aaacagatcaacgattacgtggagaagggtactcaagggaaaattgtggatttggtcaaggagcttgacagagacacagtttttgctct ggtgaattacatcttctttaaaggcaaatgggagagaccctttgaagtcaaggacaccgaggaagaggacttccacgtggaccaggtg accaccgtgaaggtgcctatgatgaagcgtttaggcatgtttaacatccagcactgtaagaagctgtccagctgggtgctgctgatgaa atacctgggcaatgccaccgccatcttcttcctgcctgatgaggggaaactacagcacctggaaaatgaactcacccacgatatcatca ccaagttcctggaaaatgaagacagaaggtctgccagcttacatttacccaaactgtccattactggaacctatgatctgaagagcgtcc tgggtcaactgggcatcactaaggtcttcagcaatggggctgacctctccggggtcacagaggaggcacccctgaagctctccaagg ccgtgcataaggctgtgctgaccatcgacgagaaagggactgaagctgctggggccatgtttttagaggccatacccatgtctatcccc cccgaggtcaagttcaacaaaccctttgtcttcttaatgattgaacaaaataccaagtctcccctcttcatgggaaaagtggtgaatccca cccaaaaataacaaagggcgaattcgtttaaacctgcaggactagcgagctcgctgatcagcctcgactgtgccttctagttgccagcc atctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgc attgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatg ctggggagagatcgatctgaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcga ccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggcccccccccc cccccccccggcgattctcttgtttgctccagactctcaggcaatgacctgatagcctttgtagagacctctcaaaaatagctaccctctc cggcatgaatttatcagctagaacggttgaatatcatattgatggtgatttgactgtctccggcctttctcacccgtttgaatctttacctaca cattactcaggcattgcatttaaaatatatgagggttctaaaaatttttatccttgcgttgaaataaaggcttctcccgcaaaagtattacagg gtcataatgtttttggtacaaccgatttagctttatgctctgaggctttattgcttaattttgctaattctttgccttgcctgtatgatttattggatg ttggaatcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgc cgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagaca agctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacg cctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttat ttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattc aacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctga agatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttcc aatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacacta ttctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccat aaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggg gatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagca atggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataa agttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtat cattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaat agacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaactt catttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcaga ccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg gtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagt gtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccag tggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtg cacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaag ggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctg gtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataacc gtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaaga gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctgattctaacgaggaaagcacgttatacgtgctc gtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgc cagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctcc ctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgata gacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctat tcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatatta acgcttacaatttaaatatttgcttatacaatcttcctgtttttggggcttttctgattatcaaccggggtacatatgattgacatgctagttttac gattaccgttcatcgcc pAAV-hCD68-RFP_pLann (SEQ ID NO: 60) tagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcg agcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtag ccatgctctttaacccgccatgctacttatctacgtagccatgctctagacactagttggtcacaagcccctcacatgcctcagccaagag gaagtagtacaggggtcagcccagaggtccaggggaaaggagtggaaaccgatttccccaccaagggaggggcctgtacctcagc tgttcccatagctacttgccacaactgccaagcaagtttcgctgagtttgacacatggatccctgtggatcaactgccctaggactccgttt gcacccatgtgacactgttgactttgccctgacgaagcagggccaacagtcccctaacttaattacaaaaactaatgactaagagagag gtggctagagctgaggcccctgagtcaggctgtgggtgggatcatctccagtacaggaagtgagactttcatttcctcctttccaagag agggctgagggagcagggttgagcaactggtgcagacagcctagctggactttgggtgaggcggttcagccatatcgaattctgctg gggctactggcaggtaaggaggaaggaggctgaggggagggggcccctgggagggagcctgccctgggttgctaaccatctcct ctctgccaaaagcccaggggacactagtgaagcttaaagtcgacaaaagacccaagctggctagccaccatgggcggcggcggct atggcaggaagaagcggagacagcgacgaagagtgcgctcctccaagaacgtcatcaaggagttcatgcgcttcaaggtgcgcatg gagggcaccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggccacaacaccgtgaagct gaaggtgaccaagggcggccccctgcccttcgcctgggacatcctgtccccccagttccagtacggctccaaggtgtacgtgaagca ccccgccgacatccccgactacaagaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcg tggtgaccgtgacccaggactcctccctgcaggacggctgcttcatctacaaggtgaagttcatcggcgtgaacttcccctccgacgg ccccgtaatgcagaagaagaccatgggctgggaggcctccaccgagcgcctgtacccccgcgacggcgtgctgaagggcgagat ccacaaggccctgaagctgaaggacggcggccactacctggtggagttcaagtccatctacatggccaagaagcccgtgcagctgc ccggctactactacgtggactccaagctggacatcacctcccacaacgaggactacaccatcgtggagcagtmmgagcgcaccga gggccgccaccacctgttcctgtagtttaagcttgggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactaccta cagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattcacagtcc caaggctcatttcaggcccctcagtcctcacagtctgttcatgatcataatcagccataccacatttgtagaggttttacttgctttaaaaaa cctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaag caatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggat ccactagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctc tctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgag cgagcgcgcagagctttttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaatagctcagaggccgaggcggcct cggcctctgcataaataaaaaaaattagtcagccatggggcggagaatgggcggaactgggcggagttaggggcgggatgggcgg agttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacct ggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacaccctaactgacacacattccac agctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgc tcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaag aacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgac gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctc cctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctca cgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcctt atccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcg aggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctg aagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcag cagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaa gggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagta aacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgt cgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccaga tttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttg ccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgttt ggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggt cctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgta agatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatac gggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccg ctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaac aggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcat ttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaag tgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggt gatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccg tcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccattc gacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtg catgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccga agtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacg atgcgtccggcgtagaggatctggctagcgatgaccctgctgattggttcgctgaccatttccgggtgcgggacggcgttaccagaaa ctcagaaggttcgtccaaccaaaccgactctgacggcagtttacgagagagatgatagggtctgcttcagtaagccagatgctacaca attaggcttgtacatattgtcgttagaacgcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactatagaata cacggaattaattc pTR2-CB-plO-microRNA-AAT (SEQ ID NO: 61)

[0155] GGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG

[0156] AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG

[0157] TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0158] AGATCTTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCA

[0159] ATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTAT

[0160] ATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTA

[0161] ATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT

[0162] ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT

[0163] TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

[0164] ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTG

[0165] TATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT

[0166] GGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTA

[0167] CGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACT

[0168] CTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTA

[0169] TTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGC

[0170] GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCA

[0171] ATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGC

[0172] GGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTT

[0173] CGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGA

[0174] CCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAA

[0175] TTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTG

[0176] AGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGC

[0177] GTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTG

[0178] AGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGA

[0179] GCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAA

[0180] GGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCG

[0181] GTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCG GCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGC

[0182] GGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGG

[0183] GGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAG

[0184] GCGCGGCGAGCCGCCGCCATTGCCTTTTATGGTAGCTAGCACCATCGACAAGAA

[0185] AGGGATTCAAGAGATCCCTTTCTTGTCGATGGTGAATTCTCAGCAATTCGATATC

[0186] AAGCTTGGGGATTTTCAGGCACCACCACTGACCTGGGACAGTGAATCGACAATG

[0187] CCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCC

[0188] TGTCTCCCTGGCTGAGGATCCCCAGGGAGATGCTGCCCAGAAGACAGATACATC

[0189] CCACCATGATCAGGATCACCCAACCTTCAACAAGATCACCCCCAACCTGGCTGA

[0190] GTTCGCCTTCAGCCTATACCGCCAGCTGGCACACCAGTCCAACAGCACCAATATC

[0191] TTCTTCTCCCCAGTGAGCATCGCTACAGCCTTTGCAATGCTCTCCCTGGGGACCA

[0192] AGGCTGACACTCACGATGAAATCCTGGAGGGCCTGAATTTCAACCTCACGGAGA

[0193] TTCCGGAGGCTCAGATCCATGAAGGCTTCCAGGAACTCCTCCGTACCCTCAACCA

[0194] GCCAGACAGCCAGCTCCAGCTGACCACCGGCAATGGCCTGTTCCTCAGCGAGGG

[0195] CCTGAAGCTAGTGGATAAGTTTTTGGAGGATGTTAAAAAGTTGTACCACTCAGAA

[0196] GCCTTCACTGTCAACTTCGGGGACACCGAAGAGGCCAAGAAACAGATCAACGAT

[0197] TACGTGGAGAAGGGTACTCAAGGGAAAATTGTGGATTTGGTCAAGGAGCTTGAC

[0198] AGAGACACAGTTTTTGCTCTGGTGAATTACATCTTCTTTAAAGGCAAATGGGAGA

[0199] GACCCTTTGAAGTCAAGGACACCGAGGAAGAGGACTTCCACGTGGACCAGGTGA

[0200] CCACCGTGAAGGTGCCTATGATGAAGCGTTTAGGCATGTTTAACATCCAGCACTG

[0201] TAAGAAGCTGTCCAGCTGGGTGCTGCTGATGAAATACCTGGGCAATGCCACCGC

[0202] CATCTTCTTCCTGCCTGATGAGGGGAAACTACAGCACCTGGAAAATGAACTCACC

[0203] CACGATATCATCACCAAGTTCCTGGAAAATGAAGACAGAAGGTCTGCCAGCTTA

[0204] CATTTACCCAAACTGTCCATTACTGGAACCTATGATCTGAAGAGCGTCCTGGGTC

[0205] AACTGGGCATCACTAAGGTCTTCAGCAATGGGGCTGACCTCTCCGGGGTCACAG

[0206] AGGAGGCACCCCTGAAGCTCTCCAAGGCCGTGCATAAGGCTGTGCTGACCATCG

[0207] ACGAGAAAGGGACTGAAGCTGCTGGGGCCATGTTTTTAGAGGCCATACCCATGT

[0208] CTATCCCCCCCGAGGTCAAGTTCAACAAACCCTTTGTCTTCTTAATGATTGAACA

[0209] AAATACCAAGTCTCCCCTCTTCATGGGAAAAGTGGTGAATCCCACCCAAAAATA

[0210] ACTGCCTCTCGCTCCTCAACCCCTCCCCTCCATCCCTGGCCCCCTCCCTGGATGAC

[0211] ATTAAAGAAGGGTTGAGCTGGTAACCCCCCCCCCCCCTGCAGGGGCCCTCGACC

[0212] CGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAAC

[0213] CACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATT

[0214] GCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCA TTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTA

[0215] AAACCTCTACAAATGTGGTAAAATCGATAAGGATCTAGGAACCCCTAGTGATGG

[0216] AGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA

[0217] GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCG

[0218] CAGAGAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCTGGCGTAATAG

[0219] CGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGTAGCCTGAATGGCGA

[0220] ATGGCGCGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTAC

[0221] GCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTC

[0222] TTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG

[0223] GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT

[0224] GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCC

[0225] CTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAAC

[0226] AACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTT

[0227] CGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAA

[0228] CAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTG

[0229] CGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCA

[0230] TAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT

[0231] TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCA

[0232] TGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCG

[0233] TGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCA

[0234] GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAAT

[0235] ACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA

[0236] TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCT

[0237] TTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTA

[0238] AAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTC

[0239] AACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGA

[0240] GCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCA

[0241] AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

[0242] CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGT

[0243] GCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCG

[0244] GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTC

[0245] GCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTG

[0246] ACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCG

[0247] AACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATA AAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA

[0248] TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCC

[0249] AGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAAC

[0250] TATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCA

[0251] TTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTC

[0252] ATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAA

[0253] AATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC

[0254] AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA

[0255] AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT

[0256] TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAG

[0257] TGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCT

[0258] CGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT

[0259] ACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA

[0260] ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTG

[0261] AGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAG

[0262] GCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA

[0263] GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC

[0264] TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA

[0265] ACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCAC

[0266] ATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA

[0267] GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG

[0268] CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC

[0269] GATTCATTAATGCAGGGCTGCAG pTR2-U6-plO-siRNA-TK-Neo (SEQ ID NO: 62)

[0270] GGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA

[0271] GGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCG

[0272] CAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTGAATTCCCCA

[0273] GTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGAGCGTGACCGCGCGCC

[0274] GAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCAT

[0275] ATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGT

[0276] AAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGG

[0277] GTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAA

[0278] CTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGCGGGAT CCACCATCGACAAGAAAGGGATTCAAGAGATCCCTTTCTTGTCGATGGTTTTTTT

[0279] GGAAAAGCTTGGGGACGCGGGTACCGCGGCCGCACTAGTCTCGAGCAGTGTGGT

[0280] TTTCAAGAGGAAGCAAAAAGCCTCTCCACCCAGGCCTGGAATGTTTCCACCCAAT

[0281] GTCGAGCAGTGTGGTTTTGCAAGAGGAAGCAAAAAGCCTCTCCACCCAGGCCTG

[0282] GAATGTTTCCACCCAATGTCGAGCAAACCCCGCCCAGCGTCTTGTCATTGGCGAA

[0283] TTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCCAGGTCCACTTCGCATATT

[0284] AAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCCAATATGGGATC

[0285] GGCCATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAG

[0286] GCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTG

[0287] TTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCG

[0288] GTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGA

[0289] CGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACT

[0290] GGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCC

[0291] TGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGAT

[0292] CCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGT

[0293] ACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAG

[0294] GGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGC

[0295] GAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAA

[0296] ATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTA

[0297] TCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATG

[0298] GGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATC

[0299] GCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCAACTCGAGAGAGCTC

[0300] GCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCC

[0301] CCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA

[0302] ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGG

[0303] GGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTG

[0304] GGGAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCT

[0305] CGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT

[0306] CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCC

[0307] CCCCCCCCCCCTGCAGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGC

[0308] GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT

[0309] CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCC

[0310] ACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAA

[0311] GGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA

[0312] GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG

[0313] TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG

[0314] GCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC

[0315] CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC

[0316] GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG

[0317] CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT

[0318] TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTG

[0319] CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGC

[0320] AAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC

[0321] GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC

[0322] TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG

[0323] GATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGT

[0324] ATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTA

[0325] TCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAG

[0326] ATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCG

[0327] CGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA

[0328] AGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTA

[0329] ATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT

[0330] TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCAT

[0331] TCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA

[0332] AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA

[0333] GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC

[0334] CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAG

[0335] TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGC

[0336] CACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAA

[0337] AACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC

[0338] ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA

[0339] ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG

[0340] AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC

[0341] TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC

[0342] GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATG

[0343] ACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCG

[0344] GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTT GTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGA

[0345] GAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACC GCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATC AAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCC ACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGG CGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGC CGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCG CCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTACG CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGGCTGCA GGGGGGGGGGGGGGGG pTR2-U6-siRNA-NC (SEQ ID NO: 63) ggttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcg gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctagatctgaattccccagtggaaaga cgcgcaggcaaaacgcaccacgtgacggagcgtgaccgcgcgccgagcgcgcgccaaggtcgggcaggaagagggcctatttc ccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaa atacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaag tatttcgatttcttgggtttatatatcttgtggaaaggacgcgggatccactaccgttgttataggtgttcaagagacacctataacaacggt agttttttggaaaagcttggggacgcgggtaccgcggccgcactagtctcgagcagtgtggttttgcaagaggaagcaaaaagcctct ccacccaggcctggaatgtttccacccaagtcgaaggcagtgtggttttgcaagaggaagcaaaaagcctctccacccaggcctgga atgtttccacccaatgtcgagcaaccccgcccagcgtcttgtcattggcgaattcgaacacgcagatgcagtcggggcggcgcggtc ccaggtccacttcgcatattaaggtgacgcgtgtggcctcgaacaccgagcgaccctgcagccaatatgggatcggccattgaacaa gatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatg ccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacga ggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggc tgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcgg cggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagcc ggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcc cgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgact gtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctg accgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaggggatcaa ctcgagagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctgg aaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggt ggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggagagatctaggaacccctagtgatggagttg gccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctc agtgagcgagcgagcgcgcagagagggagtggccaacccccccccccccccccctgcagccctgcattaatgaatcggccaacg cgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcg gtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagca aaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctc aagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgacc ctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgta ggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagag ttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaaga gttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagg atctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaa aaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatg cttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacggg agggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccag ccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagt tcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggt tcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaag ttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagt actcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagc agaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaac ccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaa agggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcg gatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaacca ttattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacac atgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttg gcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatg cgtaaggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaacca ataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccac tattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaa gttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcga acgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaacc accacacccgccgcgcttaatgcgccgctacagggcgcgtcgcgccattcgccattcaggctacgcaactgttgggaagggcgatc ggtgcgggcctcttcgctattacgccaggctgcagggggggggggggggg

[0346] Table 2. Plasmid sequence elements.

[0347] OTHER EMBODIMENTS

[0348] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0349] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0350] EQUIVALENTS

[0351] While several inventive embodiments 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 function 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 inventive embodiments described herein. 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 inventive teachings 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 inventive embodiments 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, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0352] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0353] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0354] 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.”

[0355] 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. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. 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. 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 only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0356] 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.

[0357] 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.

[0358] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0359] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” 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. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

CLAIMSThat which is claimed is:

1. A recombinant adeno-associated virus (rAAV) vector comprising a recombinant nucleic acid comprising (i) inverted terminal repeats (ITRs); (ii) a nucleotide sequence encoding an siRNA targeting a PiZ allele of alpha- 1 antitrypsin (AAT), wherein the siRNA comprises the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16; and (iii) a nucleotide sequence encoding PiM-AAT.

2. The rAAV vector of claim 1, wherein the recombinant nucleic acid comprises, from 5’ to 3’, a 5’ ITR, an enhancer, a promoter, the nucleotide sequence encoding the siRNA inserted into an intron, the nucleotide sequence encoding PiM-AAT, and a 3’ ITR.

3. The rAAV vector of claim 2, wherein the intron is (i) a chicken beta- actin intron donor of a chimeric CMV / chicken beta actin (CAG) promoter, or (ii) a SV40 intron donor.

4. The rAAV vector of claim 2 or 3, wherein the promoter is a liver specific promoter operably linked to the nucleotide sequence encoding the siRNA and / or the nucleotide sequence encoding PiM-AAT.

5. The rAAV vector of claim 2 or 3, wherein the promoter is a lung specific promoter operably linked to the nucleotide sequence encoding the siRNA and / or the nucleotide sequence encoding PiM-AAT.

6. The rAAV vector of claim 2 or 3, wherein the promoter is a macrophage specific promoter operably linked to the nucleotide sequence encoding the siRNA and / or the nucleotide sequence encoding PiM-AAT.

7. The rAAV vector of claim 6, wherein the macrophage specific promoter is a human CD68 promoter.

8. The rAAV vector of claim 7, wherein the human CD68 promoter is a truncated human CD68 promoter.

9. The rAAV vector of any one of claims 1-8, wherein the recombinant nucleic acid is self-complementary.

10. An rAAV particle comprising the rAAV vector of any one of claims 1-9.

11. The rAAV particle of claim 10, wherein the rAAV particle targets liver cells.

12. The rAAV particle of claim 10, wherein the rAAV particle targets lung cells.

13. The rAAV particle of claim 10, wherein the rAAV particle targets macrophages.

14. The rAAV particle of claim 10 or 11, wherein the rAAV particle is of serotypeAAV3.

15. The rAAV particle of any one of claims 10, 12, or 13, wherein the rAAV particle has a bovine AAV (BAAV) capsid.

16. A composition comprising a plurality of the rAAV particles of any one of claims 10- 15.

17. The composition of claim 16, further comprising a pharmaceutically acceptable carrier.

18. A method of administering the rAAV vector of any one of claims 1-9, the rAAV particle of any one of claims 10-15, or the composition of claim 16 or 17 to a subject.

19. A method of treating Alpha- 1 antitrypsin deficiency (Al AD) in a subject in need thereof, comprising administering to the subject the rAAV vector of any one of claims 1-9, the rAAV particle of any one of claims 10-15, or the composition of claim 16 or 17.

20. The method of claim 19, wherein the subject is human.

21. A variant bovine adeno-associated virus (BAAV) capsid protein comprising one or more of the following insertions in SEQ ID NO: 25:(i) a VAAA-X insertion at position 45 of SEQ ID NO: 25, wherein X is L or S; and(ii) a GAAA-X insertion at position 15 of SEQ ID NO: 25, wherein X is A or T.

22. The variant BAAV capsid protein of claim 21, comprising a VAAAL insertion at position 45 of SEQ ID NO: 25.

23. The variant BAAV capsid protein of claim 21, comprising a VAAAS insertion at position 45 of SEQ ID NO: 25.

24. The variant BAAV capsid protein of claim 21, comprising a GAAAA insertion at position 15 of SEQ ID NO: 25.

25. The variant BAAV capsid protein of claim 21, comprising a GAAAT insertion at position 15 of SEQ ID NO: 25.

26. A recombinant BAAV (rBAAV) particle comprising the variant BAAV capsid protein of any one of claims 21-25.

27. The rBAAV particle of claim 26, wherein the rBAAV particle has enhanced tropism and / or transduction efficiency for a macrophage compared to an rBAAV particle comprising a capsid protein having the amino acid sequence set forth in SEQ ID NO: 25.

28. The rBAAV particle of claim 26 or 27, further comprising a recombinant nucleic acid comprising inverted terminal repeats (ITRs).

29. The rBAAV particle of claim 28, wherein the recombinant nucleic acid comprises a nucleotide sequence encoding an siRNA targeting a PiZ allele of alpha- 1 antitrypsin (AAT).

30. The rBAAV particle of claim 29, wherein the siRNA targeting the PiZ allele of AAT comprises the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.

31. The rBAAV particle of any one of claims 28-30, wherein the recombinant nucleic acid further comprises a nucleotide sequence encoding PiM-AAT.

32. The rBAAV particle of any one of claims 28-31, wherein the recombinant nucleic acid comprises, from 5’ to 3’, a 5’ ITR, an enhancer, a promoter, the nucleotide sequence encoding the siRNA inserted into an intron, the nucleotide sequence encoding PiM-AAT, and a 3’ ITR.

33. The rBAAV particle of any one of claims 28-32, wherein the recombinant nucleic acid further comprises a macrophage specific promoter.

34. The rAAV vector of claim 33, wherein the macrophage specific promoter is a human CD68 promoter.

35. The rAAV vector of claim 34, wherein the human CD68 promoter is a truncated human CD68 promoter.

36. The rBAAV particle of any one of claims 28-35, wherein the recombinant nucleic acid is self-complementary.

37. A composition comprising a plurality of the rBAAV particles of any one of claims 26- 36.

38. The composition of claim 37, further comprising a pharmaceutically acceptable carrier.

39. A method of administering the rBAAV particle of any one of claims 26-36 or the composition of claim 37 or 38 to a subject.

40. A method of transducing a macrophage, the method comprising providing to the macrophage the rBAAV of any one of claims 26-36 or the composition of claim 37 or 38.

41. A method of treating Alpha- 1 antitrypsin deficiency (Al AD) in a subject in need thereof, comprising administering to the subject the rBAAV of any one of claims 26-36 or the composition of claim 37 or 3842. The method of claim 41, wherein the subject is human.

43. An isolated nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 20.

44. The isolated nucleic acid of claim 43, wherein the nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 20 is operably linked to (ii) a nucleotide sequence encoding an siRNA targeting the PiZ allele of alpha- 1 antitrypsin (AAT), wherein the siRNA comprises the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16; and / or (iii) a nucleotide sequence encoding PiM-AAT.

45. A vector comprising the isolated nucleic acid of claim 43 or 44.

46. The vector of claim 45, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

47. An rAAV particle comprising the rAAV vector of claim 45 or 46 encapsidated in an AAV capsid.

48. The rAAV particle of claim 47 wherein the AAV capsid comprises a capsid protein derived from bovine AAV (BAAV).

49. A composition comprising the rAAV particle of claim 47 or 48.

50. A method of treating Alpha- 1 antitrypsin deficiency (Al AD) in a subject in need thereof, comprising administering to the subject the isolated nucleic acid of claim 43 or 44, the vector of claim 45 or 46, the rAAV particle of claim 47 or 48, or the composition of claim

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