Adeno-associated virus vectors with modified membrane associated accessory protein and methods of use

Abstract

Provided herein according to some embodiments is a method of expressing a transgene from an adeno-associated virus (AAV) vector, the method comprising contacting a cell with an AAV vector comprising an AAV genome comprising the transgene and a mutated membrane associated accessory protein (MAAP) encoding sequence, such that MAAP expression is reduced or absent, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence. Compositions comprising the AAV vectors comprising the transgene and a mutated MAAP encoding sequence and methods of producing the AAVs are also provided.

Description

Attorney Docket No.5470.975.WO ADENO-ASSOCIATED VIRUS VECTORS WITH MODIFIED MEMBRANE ASSOCIATED ACCESSORY PROTEIN AND METHODS OF USE PRIORITY STATEMENT

[0001] The application claims the benefit of U.S. Provisional Application Serial No.63 / 654,588, filed May 31, 2024, the entire contents of which is incorporated by reference herein. GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number 116019 awarded by the National Institutes of Health. The government has certain rights in the invention. STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in XML format, entitled 5470-975WO_ST26.xml, 21,663 bytes in size, generated on May 29, 2025, and filed herewith, is hereby incorporated by reference in its entirety for its disclosures. FIELD OF THE INVENTION

[0004] The invention relates to adeno-associated virus vectors with enhanced transgene expression comprising the transgene and a mutated membrane associated accessory protein sequence, and methods of producing and using the vectors. BACKGROUND

[0005] Adeno-associated virus (AAV) is a ~4.7kb single-stranded Dependoparvovirus with AAV2 being the first discovered1. Since then, several serotypes of AAV have been described and created2. Recombinant AAV is the workhorse in the gene therapy field and has 5 FDA approvals. In particular, recombinant AAV9 occupies a prominent position due to its usefulness in the central nervous system evident by multiple on-going clinical trials and an FDA approved therapy3. Even with this prominence, some elements of recombinant AAV biology remain poorly characterized. The understanding of basic AAV biology comes from in vitro studies of AAV2 performed 50 years ago. The AAV genome contains two open reading frames, rep and cap, residing between two inverted terminal repeats (ITRs) that act as packaging beacons1. The rep gene encodes four proteins required for replication1. The cap open reading frame encodes threeAttorney Docket No.5470.975.WO structural capsid proteins (VP1, VP2, and VP3) present in an approximate ratio of 1:1:10 (VP1:VP2:VP3) in a fully assembled capsid4,5. VP3 is the predominant subunit on the exterior of the capsid and influences cellular / tissue tropism due to receptor binding accessibility. Only recently, two novel proteins, assembly-activating protein (AAP) and membrane-associated accessory protein (MAAP), were discovered within cap. AAP is an accessory protein6that helps assemble some AAV capsids7,8. MAAP is a membrane-associated ~13kDa protein9that enables AAV to associate with exosomes during virus production and consequentially vector trafficking into the production cell media10-12. While AAV2 elements such as ITRs and rep are present in today’s AAV vectors, differences among AAV serotypes beyond cellular / tissue tropism have not been dominant research questions.

[0006] Building on the founding in vitro AAV biology data, in vivo studies continue to shed light on AAV biology within a cell. Two were identified that aid in AAV cellulartransduction, AAVr13and GPR10814. However, the bulk of current AAV research still focuses on manipulating the capsid proteins, mainly VP3, to generate novel tissue / cell specificity. While VP3 manipulation is thought to be a key driver in AAV cellular tropism, VP1 and VP2 also prove important for AAV transduction. VP1 includes a phospholipase A2 (PLA2) domain involved in endosomal escape15, and VP1 / VP2 contain basic regions that serve as nuclear localization signals16,17. Also, studies have shown that VP1 residues are important for cellular transduction18. Recent in vitro studies found that serine residues in a conserved region of AAV2 VP1 / 2 that when mutated to alanines cause a decrease in mRNA levels, suggesting a role for VP1 / 2 in regulating transcription19. It was recently discovered that VP1 / VP2 can interact with the transgene promoter which alters the cellular tropism in both rat20and non-human primate CNS21. However, there remains a need in the art for additional improvements to AAV vectors including improved AAV vectors with enhanced transgene expression. SUMMARY OF THE INVENTION

[0007] The present invention is based, in part, on the development of AAV vectors with enhanced transgene expression, comprising an AAV genome comprising the transgene and a mutated membrane associated accessory protein (MAAP) sequence, such that MAAP expression is reduced or absent, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP sequence. Surprisingly,Attorney Docket No.5470.975.WO modification of the MAAP sequence has an effect on transgene expression in both cis and trans fashion.

[0008] In an aspect, a method of producing an AAV vector with enhanced transgene expression is provided, comprising preparing an AAV genome comprising the transgene and a mutated membrane associated accessory protein (MAAP) sequence, such that MAAP expression is reduced or absent, and producing an AAV vector comprising the AAV genome, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP sequence.

[0009] In another aspect, a method of producing an AAV vector with modulated transgene expression is provided, comprising preparing an AAV genome comprising the transgene and a mutated MAAP sequence, and producing an AAV vector comprising the AAV genome, wherein the level of transgene expression is modulated relative to an AAV vector comprising an AAV genome with a wild-type MAAP sequence.

[0010] In an aspect, a method of expressing a transgene from an AAV vector is provided, the method comprising contacting a cell with an AAV vector comprising an AAV genome comprising the transgene and a mutated MAAP sequence, such that MAAP expression is reduced or absent, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP sequence. The method may further comprise contacting the cell with an AAV vector comprising a wild-type MAAP sequence.

[0011] In another aspect, a method of expressing a transgene from an AAV vector is provided, the method comprising contacting a cell with an AAV vector comprising an AAV genome comprising the transgene and a mutated MAAP sequence, wherein the level of transgene expression is modulated relative to an AAV vector comprising an AAV genome with a wild-type MAAP sequence. The method may further comprise contacting the cell with an AAV vector comprising a wild-type MAAP sequence.

[0012] In a further aspect, a nucleic acid encoding an AAV genome comprising a mutated MAAP sequence is provided.

[0013] In an aspect, an AAV particle comprising a nucleic acid encoding an AAV genome comprising a mutated MAAP sequence is provided.

[0014] In another aspect, an AAV vector produced by the methods for producing an AAV vector is provided.Attorney Docket No.5470.975.WO

[0015] In an additional aspect, an AAV genome comprising a mutated MAAP sequence encoded by a nucleic acid as described herein is provided.

[0016] These and other aspects of the invention are set forth in more detail in the description of the invention below. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS.1A-1B. AAV9EU has reduced relative GFP mRNA levels and nuclear GFP copies compared to AAV9. AAV9-CBA-GFP and AAV9EU-CBA-GFP at equal titers were mixed 1:1 and infused into the rat striatum. Animals were sacrificed 2 weeks post infusion, striata resected, and nuclear DNA and RNA isolated. qPCR was used to determine relative GFP mRNA levels and nuclear transgene copies. A. The fold change (ΔΔct) in relative GFP mRNA levels compared to endogenous GAPDH assayed using qPCR Taqman expression assays performed on synthesized cDNA. Comparable amounts of RNA were used as input in cDNA synthesis and qPCR assays. Measurements were made in triplicate then averaged for each animal (n=4). B. The number of nuclear GFP copies per ng of genomic DNA were measured by Taqman assays for GFP and GAPDH. Comparable amounts of nuclear DNA were included in the assay with DNA concentration determined by GAPDH qPCR Taqman assay using a standard curve. Measurements were made in triplicate then averaged for each animal (n=4) . * p<0.05 using Mann-Whitney test.

[0018] FIG.2. AAV9E9 and AAVE99 exhibit a dominant neuronal cellular tropism in the rat striatum. AAV9E9-CBA-mCherry and AAVE99-CBA-mCherry were infused into the rat striatum, and 2 weeks later the animals were sacrificed, fixed, and the brain sliced (40µm). Immunohistochemistry for NeuN, a neuronal cell marker, was performed followed by confocal fluorescence microscopy. The co-localization of native mCherry fluorescence with NeuN (green) was determined by counting cells from at least 5 unique regions from each animal (n=3). There was ~90% co-localization with NeuN with both viruses. Representative maximum projected z- stacks images are shown.

[0019] FIGS.3A-3D. Transgene expression from AAV9 EU mutants and MAAP9 mutants co- localize with AAV9 transgene expression in cells morphologically resembling neurons in the rat striatum. Viruses were mixed 1:1 at equal titers and infused at equal amounts into the rat striatum. Animals were sacrificed 2 weeks post infusion and then the brains wereAttorney Docket No.5470.975.WO paraformaldehyde fixed and sectioned (40µm). Representative maximum projections of z-stacks captured by confocal microscopy of the native fluorescence signal are shown. A. AAV9-CBA- mCherry co-infused with AAV9 / MAAP9EU-CBA-GFP (n=3). B. AAV9-CBA-mCherry co- infused with either AAV9E9-CBA-GFP or AAVE99-CBA-GFP (n=3). C. AAV9-CBA-mCherry co-infused with either AAV9E9 / MAAP9EU-CBA-GFP or AAVE99 / MAAP9EU-CBA-GFP (n=3). D. AAV9-CBA-mCherry co-infused with AAV9 / no MAAP-CBA-GFP (n=3).

[0020] FIG.4. Altering MAAP reduces the fold change in the relative GFP mRNA levels in AAV9 mutants. AAV9-CBA-GFP (n=7), AAV9 / no MAAP-CBA-GFP (n=7) AAV9 / MAAPEU- CBA-GFP (n=3), AAVE99 / MAAP9-CBA-GFP (n=3), AAV9E9 / MAAP9-CBA-GFP (n=3), AAVE99 / MAAP9EU-CBA-GFP (n=3) and AAV9E9 / MAAP9EU-CBA-GFP (n=3) were infused at equal titers and amounts into the rat striatum. Two weeks post infusion RNA was isolated and cDNA was synthesized for GFP and GAPDH qPCR with Taqman expression assays. Each animal was run in triplicate then averaged. The relative GFP mRNA levels to GAPDH mRNA levels were compared to AAV9 and graphed as fold change. * indicates significance (P<0.05) using Wilcoxon matched pairs test.

[0021] FIG.5. Overview of AAV9 and AAV9 / no MAAP produced and harvested at 48 hours, and studied alone or mixed 1:1.

[0022] FIGS.6A-6B. AAV9 viruses have appropriate capsid subunit ratios. A. Purified AAV9 and AAV9EU run on a denaturing SDS gel stained with Coomassie. Both show an appropriate VP1:VP2: VP3 ratio. B. Purified AAV9 and AAV9 no MAAP run on a denaturing SDS gel stained with Coomassie. Both show an appropriate VP1:VP2: VP3 ratio.

[0023] FIG.7. Diagram of AAV genome with consequences depicted for the EU insert mutation. The result of inserting six glutamates at aa139 (VP1 numbering) are shown underlined within VP1, VP2, and MAAP on the AAV genome map. The asterisk indicates the approximate location of the insertions within each gene product.

[0024] FIG.8. When mixed AAV9 / no MAAP and AAV9 exhibit enhanced fluorescence intensity in the rat striatum. AAV9-CBA-mCherry was mixed 1:1 and co-infused with either AAV9-CBA-GFP (Left, italics) or AAV9 / no MAAP-CBA-GFP (Right, bold) into 6 rat striata. All viruses were of equal titer and an equal volume was infused.2 weeks post infusion fixed brains were sectioned in 40µm slices through the infusion sites and imaged at 4X in brightfield and native fluorescence using a slide scanner. The section where the needle tract transects theAttorney Docket No.5470.975.WO corpus callosum (labeled cc break) was used as a landmark and the first slice to be quantified. Sequential sections and images are shown for a representative animal.

[0025] FIG.9. AAV9 / no MAAP and AAV9 exhibit enhanced fluorescence intensity in the rat striatum when mixed. AAV9-CBA-mCherry was mixed 1:1 with either AAV9-CBA-GFP or AAV9 / no MAAP-CBA-GFP and infused at equal titers and volumes into the rat striatum.2 weeks post infusion the brains were paraformaldehyde fixed and 40µm sequential sections taken though the striatum infusion site. Representative 20X stitched z-stacks confocal images of the striatum infusion site are shown at the determined corpus callosum puncture (top panels) and 10 sections posterior to the corpus callosum puncture (bottom panels) as determined by slide scanner analysis (FIG.8). The confocal microscopy settings were the same for all imaging. Note the increase in transduced area and increase in fluorescence intensity when AAV9-CBA-mCherry and AAV9 / no MAAP-CBA-GFP are mixed together.

[0026] FIGS.10A-10B. When mixed AAV9 / no MAAP and AAV9 do not exhibit differences in nuclear transgene copies or fold change in relative transgene mRNA levels in the rat striatum. A. The nuclear GFP and mCherry transgene copies comparing AAV9-CBA-mCherry mixed 1:1 with either AAV9-CBA-GFP or AAV9 / no MAAP-CBA-GFP and co-infused into the rat striatum at equal titer and amounts. Nuclear DNA was isolated 2 weeks post infusion. The number of nuclear GFP copies per ng of genomic DNA measured by qPCR Taqman assays for GFP and GAPDH. B. AAV9-CBA-mCherry was mixed 1:1 and co-infused at equal titers and amounts into the rat striatum with either AAV9-CBA-GFP or AAV9 / no MAAP-CBA-GFP.2 weeks post infusion RNA was isolated and cDNA was synthesized for GFP, mCherry, and GAPDH qPCR with Taqman expression assays. The relative GFP mRNA levels were compared to AAV9-CBA-mCherry mixed with AAV9-CBA-GFP and graphed as fold change.

[0027] FIGS.11A-11B. AAV9-CBA-mCherry-MAAP9 exhibits reduced mCherry expression, reduced cellular transduction, and changes in mCherry cellular localization in vivo. A. Slide scanner brightfield and fluorescent images (4X) of the same tissue section were taken with different exposure times. The top image was taken with an exposure time of 41.32ms and the bottom image with an exposure time of 4.13s. B. Representative confocal (20X) max projection images of native mCherry fluorescence at infusion sites.Attorney Docket No.5470.975.WO DETAILED DESCRIPTION

[0028] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0030] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0031] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0032] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0033] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0034] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.Attorney Docket No.5470.975.WO

[0035] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0036] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0037] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0038] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0039] The term “consists essentially of” (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C-terminal ends of the recited sequence or additional nucleotides on the 5’ and / or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g., chaperone and / or isomerase activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0040] The term “modified MAAP” as used herein refers to the addition, deletion, and / or substitution of one or more amino acids from the wild-type MAAP protein. In some embodiments, modified MAAP is a modified MAAP9 protein.

[0041] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.Attorney Docket No.5470.975.WO

[0042] As used herein, a “functional” polypeptide or “functional fragment” is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g., wild-type protein or fragment thereof). In particular embodiments, the “functional” polypeptide or “functional fragment” substantially retains all of the activities possessed by the unmodified polypeptide (e.g., wild-type protein or fragment thereof). By “substantially retains” biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A “non- functional” polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as chaperone and / or isomerase activity can be measured using assays that are well known in the art and as described herein.

[0043] The term “fragment,” as applied to a peptide, will be understood to mean an amino acid sequence of reduced length relative to a reference peptide (e.g., wild-type protein) or amino acid sequence and comprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical to the reference peptide or amino acid sequence. Such a peptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 5, 10, 15, 20, 25, 30, 35, 46.50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutive amino acids of a peptide or amino acid sequence according to the invention.

[0044] The terms “polynucleotide”, “nucleic acid,” “nucleic acid molecule,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized), synthetic RNA of any sequence (e.g., chemically synthesized), nucleic acid probes and primers.Attorney Docket No.5470.975.WO A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base- pairing abilities or increased resistance to nucleases.

[0045] If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non- nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0046] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0047] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds.1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. Examples of the programs include BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0048] The term “endoplasmic reticulum signal sequence” (“ER signal sequence”) as used herein refers to any amino acid sequence that anchors a polypeptide in the ER.Attorney Docket No.5470.975.WO

[0049] The term “nuclear localization signal,” or “nuclear localization sequence,” (“NLS”) as used herein refers to an amino acid sequence that tags a protein for import into the cell nucleus by cell transport.

[0050] As used herein, the term “operably linked” means that the promoter and coding sequence are joined together in a manner that allows them to carry out their normal functions, i.e., transcription of the coding sequence is under the control of the promoter and the transcript produced is correctly translated into the desired product.

[0051] Those skilled in the art will appreciate that a variety of promoters may be used depending on the level and specific expression desired. The promoter may be constitutive or regulatable, depending on the pattern of expression desired. The promoter may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0052] The promoter can be native to the target cell or subject to be treated and / or can be native to the heterologous nucleotide sequence. The promoter is generally chosen so that it will function in the target cell(s) of interest. The promoter can optionally be a mammalian promoter. The promoter may further be constitutive or regulatable (e.g., inducible).

[0053] Promoters for nucleic acid delivery can be tissue preferred and / or -specific promoters. In some embodiments, the promoter is brain-specific or brain-preferred, spinal cord-specific or spinal cord-preferred, or muscle-specific or muscle-preferred.

[0054] The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, and viruses (bacteriophage, animal viruses, and plant viruses). In some embodiments the vector is a viral vector, optionally an adeno-associated virus (AAV) vector. Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and / or adenovirus vectors. Non-viral vectors include, but areAttorney Docket No.5470.975.WO not limited to, plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and / or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.). Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al., J. Biol. Chem.267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No.2,012,311, filed Mar.15, 1990).

[0055] As used herein, the term "adeno-associated virus" (AAV) includes but is not limited to, AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Recently, a number of putative new AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375-383; and Table 1). An AAV can be selected for tissue-specific delivery. AAV9 variants, for example AAV-PHP.B can be used for where desired to cross the blood brain barrier. AAV variants with reduced immunogenicity may also be utilized, and may comprise chimeric AAV, for example, AAV-DJ. Design strategies for AAV vectors may also be employed for delivery according to the present invention. See, e.g., Lee, et al. (2018) Adeno-associated virus (AAV) vectors: rational design strategies for capsid engineering. Curr. Opin. Biomed. Eng., 7, 58-63; see also Parambi et al., 2021 Oct 15, Mol Neurobiol. 2022; 59(1): 191-233, doi:10.1007 / s12035-021-02555-y, incorporated herein by reference in its entirety, and specifically Table 1 for teachings of viral vectors.Attorney Docket No.5470.975.WO Table 1 AAV GenBank AAV GenBank AAV GenBank Serotypes / Isolates Accession Serotypes / Isolates Accession Serotypes / Isolates Accession Number Number NumberAttorney Docket No.5470.975.WO

[0056] The term “tropism” as used herein refers to preferential but not necessarily exclusive entry of the vector (e.g., virus vector) into certain cell or tissue type(s) and / or preferential but not necessarily exclusive interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the vector contents (e.g., viral genome) in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequence(s). Those skilled in the art will appreciate that transcription of a heterologous nucleic acid sequence from the viral genome may not be initiated in the absence of trans-acting factors, e.g., for an inducible promoter or otherwise regulated nucleic acid sequence. In the case of a rAAV genome, gene expression from the viral genome may be from a stably integrated provirus and / or from a non- integrated episome, as well as any other form which the virus nucleic acid may take within the cell.

[0057] The term “tropism profile” refers to the pattern of transduction of one or more target cells, tissues and / or organs. Representative examples of chimeric AAV capsids have a tropism profile characterized by efficient transduction of cells of the central nervous system (CNS) with only low transduction of peripheral organs (see e.g., US Patent No.9,636,370 McCown et al., and US patent publication 2017 / 0360960 Gray et al.). Vectors (e.g., virus vectors, e.g., AAV capsids) expressing specific tropism profiles may be referred to as “tropic” for their tropism profile, e.g., neuro-tropic, liver-tropic, etc.

[0058] As used herein, “transduction” of a cell by a virus vector (e.g., an AAV vector) means entry of the vector into the cell and transfer of genetic material into the cell by the incorporation of nucleic acid into the virus vector and subsequent transfer into the cell via the virus vector.

[0059] The genomic sequences of various serotypes of AAV and the autonomous parvoviruses, as well as the sequences of the native ITRs, Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_001358, NC_001540, AF513851, AF513852 and AY530579; the disclosures of which are incorporated by reference herein for teaching parvovirus and AAV nucleic acid and amino acidAttorney Docket No.5470.975.WO sequences. See also, e.g., Bantel-Schaal et al., (1999) J. Virol.73: 939; Chiorini et al., (1997) J. Virol.71:6823; Chiorini et al., (1999) J. Virol.73:1309; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al., (2004) Virol.33-:375-383; Mori et al., (2004) Virol.330:375; Muramatsu et al., (1996) Virol.221:208; Ruffing et al., (1994) J. Gen. Virol.75:3385; Rutledge et al., (1998) J. Virol.72:309; Schmidt et al., (2008) J. Virol.82:8911; Shade et al., (1986) J. Virol.58:921; Srivastava et al., (1983) J. Virol.45:555; Xiao et al., (1999) J. Virol.73:3994; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No.6,156,303; the disclosures of which are incorporated by reference herein for teaching parvovirus and AAV nucleic acid and amino acid sequences. See also Table 1. An early description of the AAV1, AAV2 and AAV3 ITR sequences is provided by Xiao, X., (1996), “Characterization of Adeno-associated virus (AAV) DNA replication and integration,” Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, PA (incorporated herein it its entirety).

[0060] Unless indicated otherwise, “efficient transduction” or “efficient tropism,” or similar terms, can be determined by reference to a suitable positive or negative control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism, respectively, of a positive control or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism, respectively, of a negative control).

[0061] Similarly, it can be determined if a virus “does not efficiently transduce” or “does not have efficient tropism” for a target tissue, or similar terms, by reference to a suitable control. In particular embodiments, the virus vector does not efficiently transduce (i.e., does not have efficient tropism for) tissues outside the CNS, e.g., liver, kidney, gonads and / or germ cells. In particular embodiments, undesirable transduction of tissue(s) (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of the desired target tissue(s) (e.g., CNS cells).

[0062] The terms “5’ portion” and “3’ portion” are relative terms to define a spatial relationship between two or more elements. Thus, for example, a “3’ portion” of a polynucleotide indicates a segment of the polynucleotide that is downstream of another segment. The term “3’ portion” is not intended to indicate that the segment is necessarily at the 3’ end of the polynucleotide, or even that it is necessarily in the 3’ half of the polynucleotide, although it may be. Likewise, a “5’ portion” of a polynucleotide indicates a segment of the polynucleotide that is upstream of another segment. The term “5’ portion” is not intended to indicate that the segment is necessarilyAttorney Docket No.5470.975.WO at the 5’ end of the polynucleotide, or even that it is necessarily in the 5’ half of the polynucleotide, although it may be.

[0063] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

[0064] As used herein, the term “host cell” refers to a cell that is engineered to express the modified polypeptide or functional fragment thereof (e.g., a modified full length [protein] or a fragment thereof). “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0065] Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Prokaryotes include gram negative or positive cells. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and / or expression include DH5α, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.

[0066] “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) refers to a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that isAttorney Docket No.5470.975.WO generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term “carrier” encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0067] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0068] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0069] “Inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1, 5, 10, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0070] The term “contact” or grammatical variations thereof as used with respect to a polypeptide and a cell or aggregate, refers to bringing the polypeptide and the cell or aggregate in sufficiently close proximity to each other for one to exert a biological effect on the other. In some embodiments, the term contact means binding of the polypeptide to the cell or aggregate.

[0071] A “subject” may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, lagomorph (e.g., rabbit), or rodent (e.g., mouse, rat). In some embodiments a human subject is a neonate, child, adult or geriatric subject. In some embodiments a human subject is at least 50, 60, 70, 80, or 90 years old.Attorney Docket No.5470.975.WO

[0072] “Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0073] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds areAttorney Docket No.5470.975.WO administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0074] The present disclosure is based in part on the discovery of increased transgene mRNA levels and increased protein expression from AAV vectors with modified membrane associated accessory proteins. Compositions

[0075] In some embodiments, a nucleic acid encoding an AAV genome comprising a mutated membrane associated accessory protein (MAAP) encoding sequence is provided. The wild-type MAAP sequence from various AAV serotypes is known in the art. For example, the wild-type MAAP encoding sequence of AAV9 comprises SEQ ID NO:22. The wild-type MAAP amino acid sequence of AAV9 comprises SEQ ID NO:23. In some embodiments, the MAAP encoding sequence is from a corresponding AAV serotype. MAAP is translated from an alternate reading frame (+2 with VPs + 1) on the VP2 / VP3 / AAP transcript. The MAAP encoding sequence from a corresponding serotype is known in the art and can be utilized in accordance with this disclosure, including, without limitation, mutated MAAP encoding sequence or production of an AAV vector with no MAAP. Amino acid sequence alignment of MAAPs from AAV serotypes show conserved N- and C-terminal regions, with predicted structure of the N-terminus comprising a conserved N-terminal hydrophobic motif with both alpha-helical and beta strand secondary structure, T / S rich sequence clusters and a C-terminal domain with a hydrophobic alpha helical motif and a cluster of R / K residues. See, Fig.1B of Elmore et al., Nature Communications 12:6239 (2021) (showing sequence alignment of MAAP proteins), specifically incorporatedAttorney Docket No.5470.975.WO herein by reference. In some embodiments, an AAV vector comprising a mutated MAAP encoding sequence is provided. In some embodiments, the mutated MAAP encoding sequence can comprise a deletion, insertion, and / or substitution. In some embodiments, the mutated MAAP encoding sequence comprises a deletion of one or more codons.

[0076] In some embodiments, the MAAP encoding sequence is partially (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% deleted) or completely deleted. In some embodiments, the expression of MAAP is partially (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) or completely repressed.

[0077] In some embodiments, the mutated MAAP encoding sequence comprises an insertion encoding one or more amino acids. One or more sequence changes encoding conservative amino acid mutations can also be included in the mutated MAAP encoding sequence and / or the AAV genome. In some embodiments, the mutated MAAP encoding sequence comprises an insertion in the AAV genome encoding, 4-8 amino acids, e.g., 6 amino acids, for example, 6 glutamates or 6 alanines, resulting in the MAAP open reading frame being modified. In an embodiment, an insertion in the MAAP encoding sequence at a position encoding the C-terminal of MAAP is provided. In some embodiments, the insertion is at a position at a site corresponding to AAV9 (VP1 numbering) at amino acid 139 (aa139). In some embodiments, a mutation comprising an insertion in the nucleic acid sequence corresponding to position aa139 results in a RRKRRK (SEQ ID NO:1) insertion into the C-terminus of MAAP9.

[0078] In an embodiment, a six glutamate insertion (EU mutation) is inserted into AAV at a site corresponding to AAV9 (VP1 numbering) at amino acid 139 (aa139). In some embodiments, a mutation comprising an insertion in the nucleic acid sequence corresponding to position aa139 results in a RRKRRK (SEQ ID NO:1) insertion into the C-terminus of MAAP9.

[0079] In some embodiments an AAV vector encoding transgene mRNA is provided. In some embodiments, the insertion of six basic amino acids, e.g., glutamates or alanines, at amino acid 139 of AAV9 or at a corresponding position in another AAV serotype decreases transgene mRNA encoded in the AAV vector relative to an AAV without the insertion. In some embodiments, the insertion results in an insertion in the C-terminus of an encoded MAAP protein.

[0080] In some embodiments, the AAV is mutated to prevent or reduce expression of MAAP. In some embodiments, the mutated MAAP encoding sequence comprises a mutation in a startAttorney Docket No.5470.975.WO codon of MAAP. In some embodiments, the start codon of the MAAP sequence is mutated from CTG to CCG. In some embodiments, the start codon of the MAAP sequence is mutated to a stop codon. In some embodiments, an AAV comprising the mutated MAAP encoding sequence results in AAV with no MAAP. In some embodiments, when an AAV with no MAAP is mixed with AAV wild type or AAV encoding MAAP, the combination of AAV with no map and AAV increases transgene protein levels. In some embodiments, the mixture increases transgene mRNA levels. In some embodiments, the mixture of AAV with no MAAP and AAV with MAAP comprises a mixture of AAV of the same serotype, e.g., AAV9 with no MAAP and AAV9. In some embodiments, the ratio of the mixture of AAV and AAV / no MAAP is between 1:10 to 10:1 vector genome (vg):vg, e.g., between 1:5 to 5:1, between 1:3 to 3:1, or between 1:2 to 2:1. In some embodiments, the mixture allows for use of a reduced AAV titer while maintaining desired therapeutic levels relative to delivery of an AAV without the mutated MAAP.

[0081] In some embodiments, the AAV is mutated to increase expression of MAAP. In some embodiments, the start codon is mutated from CTG to ATG. In some embodiments MAAP is expressed with a promoter during production to increase expression. Example promoters for increasing expression of MAAP include chicken beta actin promoter or cytomegalovirus promoter.

[0082] In some embodiments, the mutated MAAP encoding sequence results in a level of MAAP expression that is less than 50%, 40%, 30%, 20%, or 10% of the level of MAAP expression from an AAV genome comprising a wild-type MAAP encoding sequence. In some embodiments, the mutated MAAP encoding sequence results in a level of MAAP expression that is at least 10%, 20%, 30%, 40%, 50%, or 100% greater than the level of MAAP expression from an AAV genome comprising a wild-type MAAP encoding sequence.

[0083] It will be apparent to those skilled in the art that the amino acid sequences of the AAV comprising a mutated MAAP sequence can further be modified to incorporate other modifications as known in the art to impart desired properties. As nonlimiting possibilities, the capsid protein can be modified to incorporate targeting sequences (e.g., RGD) or sequences that facilitate purification and / or detection. For example, the capsid protein can be fused to all or a portion of glutathione-S-transferase, maltose-binding protein, a heparin / heparan sulfate binding domain, poly-His, a ligand, and / or a reporter protein (e.g., Green Fluorescent Protein, β- glucuronidase, β-galactosidase, luciferase, etc.), an immunoglobulin Fc fragment, a single-chainAttorney Docket No.5470.975.WO antibody, hemagglutinin, c-myc, FLAG epitope, and the like to form a fusion protein. Methods of inserting targeting peptides into the AAV capsid are known in the art (see, e.g., International Patent Publication WO 00 / 28004; Nicklin et al., (2001) Mol. Ther.474-181; White et al., (2004) Circulation 109:513-319; Muller et al., (2003) Nature Biotech.21:1040-1046).

[0084] Conservative amino acid substitutions are known in the art. In particular embodiments, a conservative amino acid substitution includes substitutions within one or more of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and / or phenylalanine, tyrosine.

[0085] In some embodiments, the nucleic acid further encodes a promoter operably linked to the MAAP sequence. The promoter may be a cell specific promoter. Examples of cell specific promoters include, for example, promoters selected from the eukaryotic promoter database, available at epd.expasy.org. Those skilled in the art will appreciate that a variety of promoter / enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter / enhancer can be constitutive or inducible, depending on the pattern of expression desired. The promoter / enhancer can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0086] In particular embodiments, the promoter / enhancer elements can be native to the target cell or subject to be treated. In representative embodiments, the promoters / enhancer element can be native to the nucleic acid sequence. The promoter / enhancer element is generally chosen so that it functions in the target cell(s) of interest. Further, in particular embodiments the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element may be constitutive or inducible. Inducible expression control elements are typically advantageous in those applications in which it is desirable to provide regulation over expression of the nucleic acid sequence(s). Inducible promoters / enhancer elements for gene delivery can be tissue-specific or –preferred promoter / enhancer elements, and include neural tissue specific or preferred (including brain-specific or preferred), promoter / enhancer elements. Inducible promoters / enhancer elements for gene delivery can be tissue-specific or tissue-preferred promoter / enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and / or smooth muscle), neural tissue specific or preferred (including brain-specific), eye (including retina-specific and cornea-specific), liver specific or preferred, bone marrow specificAttorney Docket No.5470.975.WO or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter / enhancer elements. Exemplary neuronal promoters for use with AAVs include the human Synapsin promoter (hSyn1) and mouse PGK (mPGK). Examples of neuron- specific or preferred promoters include, without limitation, neuronal-specific enolase, synapsin, and MeCP2. Examples of astrocyte-specific or preferred promoters include, without limitation, glial fibrillary acidic protein (GFAP), S100β and GfaABC1D promoters. See, e.g., Merienne N, Le Douce J, Faivre E, Deglon N, Bonvento G. Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors. Griffin, J.M., Fackelmeier, B., Fong, D.M. et al. Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in robust transduction in the rat spinal cord following injury. Gene Ther 26, 198–210 (2019). CBA promoter (Chicken β-actin) Examples of ependymal cell-specific or preferred promoters include, without limitation, wdr16, Foxj1, and LRP2. Examples of microglia-specific or preferred promoters include, without limitation, F4 / 80, CX3CR1, and CD11b. Examples of oligodendrocyte-specific or preferred promoters include, without limitation, myelin basic protein, cyclic nucleotide phosphodiesterase, proteolipid protein, Gtx, and Sox10. Use of a CNS cell-specific or preferred promoter can increase the specificity achieved by the chimeric AAV vector by further limiting expression of the heterologous nucleic acid to the CNS. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements or cell stress-inducible elements. Other example promoters that aid in delivery of AAV vectors, for example, maiximize AAV2 cargo space for gene delivery, include Chicken β-actin (CBS), cytomegalovirus (CMV), short CMV early enhancer / chicken β-actin / short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). Exemplary inducible promoters / enhancer elements include, but are not limited to, a Tet on / off element, a RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter.

[0087] In embodiments wherein the nucleic acid sequence(s) is transcribed and then translated in the target cells, specific initiation signals are generally included for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the initiation codon (e.g., ATG) and adjacent sequences, can be of a variety of origins, both natural and synthetic.Attorney Docket No.5470.975.WO

[0088] In some embodiments, the nucleic acid further comprises a transgene sequence. Advantageously, in some embodiments, the present disclosure provides means for increased mRNA levels of the transgene and / or increased expression of the transgene. The transgene can encode a functional nucleic acid. In some embodiments, the transgene encodes a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide. In general, the delivery vectors of the disclosure may be employed to deliver any foreign nucleic acid, e.g., transgene, with a biological effect to treat or ameliorate the symptoms associated with any disorder related to gene expression. Further, the compositions of the invention (e.g., AAV with modified MAAP) can be used to treat any disease state for which it is beneficial to deliver a therapeutic polypeptide. In some embodiments, the polypeptide is one that stimulates growth and / or differentiation of CNS cells, e.g., neurons, glial cells, oligodendrocytes, astrocytes, microglia, and / or ependymal cells. Examples include, without limitation, insulin-like growth factor-1, glial-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, artemin, neurterin, persephin, brain-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor, transforming growth factor alpha, platelet-derived growth factor, leukemia inhibitory factor, prolactin, monocarboxylate transporter 1, or nuclear factor 1A.

[0089] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin (including the protein product of dystrophin mini-genes or micro-genes, see, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003017131; Wang et al., (2000) Proc. Natl. Acad. Sci. USA 97:13714-9 [mini-dystrophin]; Harper et al., (2002) Nature Med.8:253-61 [micro-dystrophin]); mini-agrin, a laminin-α2, a sarcoglycan (α, β, γ or δ), Fukutin-related protein, myostatin pro-peptide, follistatin, dominant negative myostatin, an angiogenic factor (e.g., VEGF, angiopoietin-1 or 2), an anti-apoptotic factor (e.g., heme-oxygenase-1, TGF-β, inhibitors of pro-apoptotic signals such as caspases, proteases, kinases, death receptors [e.g., CD-095], modulators of cytochrome C release, inhibitors of mitochondrial pore opening and swelling); activin type II soluble receptor, anti- inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin, mini- utrophin, antibodies or antibody fragments against myostatin or myostatin propeptide, cell cycle modulators, Rho kinase modulators such as Cethrin, which is a modified bacterial C3 exoenzyme [available from BioAxone Therapeutics, Inc., Saint-Lauren, Quebec, Canada], BCL-xL, BCL2, XIAP, FLICEc-s, dominant-negative caspase-8, dominant negative caspase-9, SPI-6 (see, e.g.,Attorney Docket No.5470.975.WO U.S. Patent Application No.20070026076), transcriptional factor PGC-α1, Pinch gene, ILK gene and thymosin β4 gene), clotting factors (e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, an intracellular and / or extracellular superoxide dismutase, leptin, the LDL receptor, neprilysin, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α1-antitrypsin, methyl cytosine binding protein 2, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, β- glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase, RP65 protein, a cytokine (e.g., α-interferon, β-interferon, interferon-γ, interleukins-1 through -14, granulocyte-macrophage colony stimulating factor, lymphotoxin, and the like), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors including IGF-1 and IGF-2, GLP-1, platelet derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor –3 and –4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor –α and –β, and the like), bone morphogenic proteins (including RANKL and VEGF), a lysosomal protein, a glutamate receptor, a lymphokine, soluble CD4, an Fc receptor, a T cell receptor, ApoE, ApoC, inhibitor 1 of protein phosphatase inhibitor 1 (I-1), phospholamban, serca2a, lysosomal acid α-glucosidase, α-galactosidase A, Barkct, β2-adrenergic receptor, β2- adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), calsarcin, a receptor (e.g., the tumor necrosis growth factor-α soluble receptor), an anti-inflammatory factor such as IRAP, Pim-1, PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-β4, hypoxia-inducible transcription factor [HIF], an angiogenic factor, S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effects G-protein coupled receptor kinase type 2 knockdown such as a truncated constitutively active bARKct; phospholamban inhibitory or dominant-negative molecules such as phospholamban S16E, a monoclonal antibody (including single chain monoclonal antibodies) or a suicide gene product (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factors such as TNF-α), and any other polypeptide that has a therapeutic effect in a subject in need thereof.

[0090] Nucleotide sequences encoding polypeptides include those encoding reporter polypeptides (e.g., an enzyme). Reporter polypeptides are known in the art and include, but are not limited to, a fluorescent protein (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), an enzyme that produces a detectable product, such as luciferase (e.g., from Gaussia, Renilla, or Photinus),Attorney Docket No.5470.975.WO β-galactosidase, β-glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene, or proteins that can be directly detected. Virtually any protein can be directly detected by using, for example, specific antibodies to the protein. Additional markers (and associated antibiotics) that are suitable for either positive or negative selection of eukaryotic cells are disclosed in Sambrook and Russell (2001), Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Ausubel et al. (1992), Current Protocols in Molecular Biology, John Wiley & Sons, including periodic updates.

[0091] In embodiments wherein the transgene nucleic acid sequence(s) is transcribed and then translated in the target cells, specific initiation signals are generally employed for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0092] The transgene may encode a functional RNA, e.g., an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Patent No.5,877,022), RNAs that effect spliceosome- mediated trans-splicing (see, Puttaraju et al., (1999) Nature Biotech.17:246; U.S. Patent No. 6,013,487; U.S. Patent No.6,083,702), interfering RNAs (RNAi) including small interfering RNAs (siRNA) that mediate gene silencing (see, Sharp et al., (2000) Science 287:2431), microRNA, or other non-translated “functional” RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Patent No.5,869,248 to Yuan et al.), and the like. Exemplary untranslated RNAs include RNAi or antisense RNA against the multiple drug resistance (MDR) gene product (e.g., to treat tumors and / or for administration to the heart to prevent damage by chemotherapy), RNAi or antisense RNA against myostatin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA against VEGF or a tumor immunogen including but not limited to those tumor immunogens specifically described herein (to treat tumors), RNAi or antisense oligonucleotides targeting mutated dystrophins (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA against the hepatitis B surface antigen gene (to prevent and / or treat hepatitis B infection), RNAi or antisense RNA against the HIV tat and / or rev genes (to prevent and / or treat HIV) and / or RNAi or antisense RNA against any other immunogen from a pathogen (to protect a subject from the pathogen) or a defective gene product (to prevent or treat disease). RNAi or antisense RNA against the targets described above or any other target can also be employed as a research reagent.Attorney Docket No.5470.975.WO

[0093] As is known in the art, anti-sense nucleic acids (e.g., DNA or RNA) and inhibitory RNA (e.g., microRNA and RNAi such as siRNA or shRNA) sequences can be used to induce “exon skipping” in patients with muscular dystrophy arising from defects in the dystrophin gene. Thus, the heterologous nucleic acid can encode an antisense nucleic acid or inhibitory RNA that induces appropriate exon skipping. Those skilled in the art will appreciate that the particular approach to exon skipping depends upon the nature of the underlying defect in the dystrophin gene, and numerous such strategies are known in the art. Exemplary antisense nucleic acids and inhibitory RNA sequences target the upstream branch point and / or downstream donor splice site and / or internal splicing enhancer sequence of one or more of the dystrophin exons (e.g., exons 19 or 23). For example, in particular embodiments, the heterologous nucleic acid encodes an antisense nucleic acid or inhibitory RNA directed against the upstream branch point and downstream splice donor site of exon 19 or 23 of the dystrophin gene. Such sequences can be incorporated into an AAV vector delivering a modified U7 snRNA and the antisense nucleic acid or inhibitory RNA (see, e.g., Goyenvalle et al., (2004) Science 306:1796-1799). As another strategy, a modified U1 snRNA can be incorporated into an AAV vector along with siRNA, microRNA or antisense RNA complementary to the upstream and downstream splice sites of a dystrophin exon (e.g., exon 19 or 23) (see, e.g., Denti et al., (2006) Proc. Nat. Acad. Sci.USA 103:3758-3763). Further, antisense nucleic acids and inhibitory RNA can target the splicing enhancer sequences within exons 19, 43, 45 or 53 (see, e.g., U.S. Patent No.6,653,467; U.S. Patent No.6,727,355; and U.S. Patent No.6,653,466).

[0094] Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim et al., (1987) Proc. Natl. Acad. Sci. USA 84:8788; Gerlach et al., (1987) Nature 328:802; Forster and Symons, (1987) Cell 49:211). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel and Westhof, (1990) J. Mol. Biol. 216:585; Reinhold-Hurek and Shub, (1992) Nature 357:173). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction.

[0095] Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage / ligation reactions involving nucleic acids (Joyce, (1989) Nature 338:217). ForAttorney Docket No.5470.975.WO example, U.S. Pat. No.5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of nucleic acid expression may be particularly suited to therapeutic applications (Scanlon et al., (1991) Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science 247:1222; Sioud et al., (1992) J. Mol. Biol.223:831).

[0096] MicroRNAs (mir) are natural cellular RNA molecules that can regulate the expression of multiple genes by controlling the stability of the mRNA. Over-expression or diminution of a particular microRNA can be used to treat a dysfunction and has been shown to be effective in a number of disease states and animal models of disease (see, e.g., Couzin, (2008) Science 319:1782-4). The chimeric AAV can be used to deliver microRNA into cells, tissues and subjects for the treatment of genetic and acquired diseases, or to enhance functionality and promote growth of certain tissues. For example, mir-1, mir-133, mir-206 and / or mir-208 can be used to treat cardiac and skeletal muscle disease (see, e.g., Chen et al., (2006) Genet.38:228-33; van Rooij et al., (2008) Trends Genet.24:159-66). MicroRNA can also be used to modulate the immune system after gene delivery (Brown et al., (2007) Blood 110:4144-52).

[0097] The term “antisense oligonucleotide” (including “antisense RNA”) as used herein, refers to a nucleic acid that is complementary to and specifically hybridizes to a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids that encode the same can be made in accordance with conventional techniques. See, e.g., U.S. Patent No.5,023,243 to Tullis; U.S. Patent No.5,149,797 to Pederson et al.

[0098] Those skilled in the art will appreciate that it is not necessary that the antisense oligonucleotide be fully complementary to the target sequence as long as the degree of sequence similarity is sufficient for the antisense nucleotide sequence to specifically hybridize to its target (as defined above) and reduce production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

[0099] To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be carried out under conditions of reduced stringency, medium stringency or even stringent conditions. Suitable conditions for achieving reduced, medium and stringent hybridization conditions are as described herein.Attorney Docket No.5470.975.WO

[0100] Alternatively stated, in particular embodiments, antisense oligonucleotides of the invention have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity with the complement of the target sequence and reduce production of the protein product (as defined above). In some embodiments, the antisense sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the target sequence. Methods of determining percent identity of nucleic acid sequences are described in more detail elsewhere herein.

[0101] The length of the antisense oligonucleotide is not critical as long as it specifically hybridizes to the intended target and reduces production of the protein product (as defined above) and can be determined in accordance with routine procedures. In general, the antisense oligonucleotide is at least about eight, ten or twelve or fifteen nucleotides in length and / or less than about 20, 30, 40, 50, 60, 70, 80, 100 or 150 nucleotides in length.

[0102] RNA interference (RNAi) is another useful approach for reducing production of a protein product (e.g., shRNA or siRNA). RNAi is a mechanism of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) corresponding to a target sequence of interest is introduced into a cell or an organism, resulting in degradation of the corresponding mRNA. The mechanism by which RNAi achieves gene silencing has been reviewed in Sharp et al., (2001) Genes Dev 15: 485-490; and Hammond et al., (2001) Nature Rev. Gen.2:110-119). The RNAi effect persists for multiple cell divisions before gene expression is regained. RNAi is therefore a powerful method for making targeted knockouts or “knockdowns” at the RNA level. RNAi has proven successful in human cells, including human embryonic kidney and HeLa cells (see, e.g., Elbashir et al., Nature (2001) 411:494-8).

[0103] Initial attempts to use RNAi in mammalian cells resulted in antiviral defense mechanisms involving PKR in response to the dsRNA molecules (see, e.g., Gil et al., (2000) Apoptosis 5:107). It has since been demonstrated that short synthetic dsRNA of about 21 nucleotides, known as “short interfering RNAs” (siRNA) can mediate silencing in mammalian cells without triggering the antiviral response (see, e.g., Elbashir et al., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad. Sci. USA 98:9742).

[0104] The RNAi molecule (including an siRNA molecule) can be a short hairpin RNA (shRNA; see Paddison et al., (2002), Proc. Nat. Acad. Sci. USA 99:1443-1448), which is believed to be processed in the cell by the action of the RNase III like enzyme Dicer into 20- 25mer siRNA molecules. The shRNAs generally have a stem-loop structure in which twoAttorney Docket No.5470.975.WO inverted repeat sequences are separated by a short spacer sequence that loops out. There have been reports of shRNAs with loops ranging from 3 to 23 nucleotides in length. The loop sequence is generally not critical. Exemplary loop sequences include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA.

[0105] The RNAi can further comprise a circular molecule comprising sense and antisense regions with two loop regions on either side to form a “dumbbell” shaped structure upon dsRNA formation between the sense and antisense regions. This molecule can be processed in vitro or in vivo to release the dsRNA portion, e.g., a siRNA.

[0106] International patent publication WO 01 / 77350 describes a vector for bi-directional transcription to generate both sense and antisense transcripts of a heterologous sequence in a eukaryotic cell. This technique can be employed to produce RNAi for use according to the invention.

[0107] Shinagawa et al., (2003) Genes Dev.17:1340 reported a method of expressing long dsRNAs from a CMV promoter (a pol II promoter), which method is also applicable to tissue specific pol II promoters. Likewise, the approach of Xia et al., (2002) Nature Biotech.20:1006, avoids poly(A) tailing and can be used in connection with tissue-specific promoters.

[0108] Methods of generating RNAi include chemical synthesis, in vitro transcription, digestion of long dsRNA by Dicer (in vitro or in vivo), expression in vivo from a delivery vector, and expression in vivo from a PCR-derived RNAi expression cassette (see, e.g., TechNotes 10(3) “Five Ways to Produce siRNAs,” from Ambion, Inc., Austin TX; available at www.ambion.com).

[0109] Guidelines for designing siRNA molecules are available (see e.g., literature from Ambion, Inc., Austin TX; available at www.ambion.com). In particular embodiments, the siRNA sequence has about 30-50% G / C content. Further, long stretches of greater than four T or A residues are generally avoided if RNA polymerase III is used to transcribe the RNA. Online siRNA target finders are available, e.g., from Ambion, Inc. (www.ambion.com), through the Whitehead Institute of Biomedical Research (jura.wi.mit.edu) or from Dharmacon Research, Inc. (dharmacon.com).

[0110] The antisense region of the RNAi molecule can be completely complementary to the target sequence, but need not be as long as it specifically hybridizes to the target sequence (as defined above) and reduces production of the protein product (e.g., by at least about 30%, 40%,Attorney Docket No.5470.975.WO 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, hybridization of such oligonucleotides to target sequences can be carried out under conditions of reduced stringency, medium stringency or even stringent conditions, as defined above.

[0111] In other embodiments, the antisense region of the RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity with the complement of the target sequence and reduces production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the antisense region contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the target sequence. Mismatches are generally tolerated better at the ends of the dsRNA than in the center portion.

[0112] In particular embodiments, the RNAi is formed by intermolecular complexing between two separate sense and antisense molecules. The RNAi comprises a ds region formed by the intermolecular basepairing between the two separate strands. In other embodiments, the RNAi comprises a ds region formed by intramolecular basepairing within a single nucleic acid molecule comprising both sense and antisense regions, typically as an inverted repeat (e.g., a shRNA or other stem loop structure, or a circular RNAi molecule). The RNAi can further comprise a spacer region between the sense and antisense regions.

[0113] Generally, RNAi molecules are highly selective. If desired, those skilled in the art can readily eliminate candidate RNAi that are likely to interfere with expression of nucleic acids other than the target by searching relevant databases to identify RNAi sequences that do not have substantial sequence homology with other known sequences, for example, using BLAST (available at www.ncbi.nlm.nih.gov / BLAST).

[0114] Kits for the production of RNAi are commercially available, e.g., from New England Biolabs, Inc. and Ambion, Inc.

[0115] The recombinant virus vector may also comprise a heterologous nucleotide sequence that shares homology with and recombines with a locus on the host chromosome. This approach may be utilized to correct a genetic defect in the host cell.

[0116] The disclosure also provides AAV particles comprising an AAV capsid and an AAV genome, wherein the AAV genome “corresponds to” (i.e., encodes) the AAV capsid. Also provided are collections or libraries of such chimeric AAV particles, wherein the collection or library comprises 2 or more, 10 or more, 50 or more, 100 or more, 1000 or more, 104or more, 105or more, or 106or more distinct sequences.Attorney Docket No.5470.975.WO

[0117] The present invention further encompasses “empty” capsid particles (i.e., in the absence of a vector genome) comprising, consisting of, or consisting essentially of the chimeric AAV capsid proteins of the invention. The chimeric AAV capsids of the invention can be used as “capsid vehicles,” as has been described in U.S. Patent No.5,863,541. Molecules that can be covalently linked, bound to or packaged by the virus capsids and transferred into a cell include DNA, RNA, a lipid, a carbohydrate, a polypeptide, a small organic molecule, or combinations of the same. Further, molecules can be associated with (e.g., “tethered to”) the outside of the virus capsid for transfer of the molecules into host target cells. In one embodiment of the invention the molecule is covalently linked (i.e., conjugated or chemically coupled) to the capsid proteins. Methods of covalently linking molecules are known by those skilled in the art.

[0118] In some embodiments, the AAV vector comprises a capsid protein having a tissue- specific tropism, for example to CNS. Thus, one aspect of the invention relates to a method of preparing an AAV capsid having a tropism profile of interest, the method comprising modifying the AAV capsid of the present invention to insert an amino acid sequence providing the tropism profile of interest. In some embodiments, the tropism profile of interest is enhanced selectivity for a tissue selected from skeletal muscle, cardiac muscle, diaphragm, kidney, liver, pancreas, spleen, gastrointestinal tract, lung, joint tissue, tongue, ovary, testis, a germ cell, a cancer cell, or a combination thereof and / or reduced selectivity for a tissue selected from liver, ovary, testis, a germ cell, or a combination thereof.

[0119] Examples of specific targeting and detargeting sequences are known in the art. One example is the molecular basis for preferential liver tropism, which has been mapped, in the case of AAV2 and AAV6, to a continuous basic footprint that appears to be involved in the interaction of either serotype with heparin. Specifically, it has previously been demonstrated that a single lysine residue on AAV6 (K531) dictates heparin binding ability and consequently, liver tropism. In corollary, substitutional mutagenesis of the corresponding glutamate / aspartate residue on other serotypes with a lysine residue confers heparin binding, possibly by forming a minimum continuous basic footprint on the capsid surface. Another example is the capsid mutants comprising alterations in the three-fold axis loop 4 as disclosed in International Publication No. WO 2012 / 093784, incorporated herein by reference in its entirety. These mutants exhibit one or more properties including (i) reduced transduction of liver, (ii) enhanced movement across endothelial cells, (iii) systemic transduction; (iv) enhanced transduction ofAttorney Docket No.5470.975.WO muscle tissue (e.g., skeletal muscle, cardiac muscle and / or diaphragm muscle), and / or (v) reduced transduction of brain tissues (e.g., neurons). Other tropic sequences are described in Li et al., (2012) J. Virol.86:7752-7759; Pulicherla et al., (2011) Mol. Ther.19:1070-1078; Bowles et al., (2012) Mol. Ther.20:443-455; Asokan et al., (2012) Mol. Ther.20:699-708; and Asokan et al., (2010) Nature Biotechnol.28:79-82; each incorporated by reference in its entirety. In some embodiments, the AAV capsid of the present invention can be modified through DNA scrambling and / or directed evolution to identify modified capsids having the desired tropism profile. Techniques for DNA scrambling and directed evolution of AAV capsids are described in International Publication No. WO 2009 / 137006, incorporated herein by reference in its entirety.

[0120] In some embodiments, the AAV vector is an AAV1, AAV2, AAV5 or AAV9 serotype.

[0121] In some embodiments, the nucleic acid is a plasmid, phage, viral vector (e.g., AAV vector, an adenovirus vector, a herpesvirus vector, or a baculovirus vector), bacterial artificial chromosome, or yeast artificial chromosome. In some embodiments, the nucleic acid is an AAV vector comprising the coding sequence.

[0122] The nucleic acid can be incorporated into a delivery vector, such as a viral delivery vector. To illustrate, the nucleic acid of the invention can be packaged in an AAV particle, an adenovirus particle, a herpesvirus particle, a baculovirus particle, or any other suitable virus particle. In some embodiments, an AAV particle comprising a nucleic acid encoding an AAV genome comprising a mutated MAAP encoding sequence is provided. In some embodiments, an AAV vector produced by the methods for producing an AAV vector is provided. In some embodiments, an AAV genome comprising a mutated MAAP encoding sequence encoded by a nucleic acid as described herein is provided.

[0123] The cell(s) into which the delivery vector is introduced can be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons and oligodendrocytes), lung cells, cells of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), blood vessel cells (e.g., endothelial cells, intimal cells), epithelial cells (e.g., gut and respiratory epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells and / or diaphragm muscle cells), dendritic cells, endothelial cells, germ cells, and the like. In representative embodiments, the cell can be any progenitor cell. As a further possibility, the cell can be a stemAttorney Docket No.5470.975.WO cell (e.g., neural stem cell, liver stem cell). Moreover, the cell can be from any species of origin, as indicated above. Furthermore, the cells may be dividing or non-dividing.

[0124] Embodiments of the invention may be performed in vitro or in vivo. One aspect of the present invention is a method of expressing a transgene in a cell in vitro, e.g., for research purposes or as part of an ex vivo method. The transgene delivery vector may be introduced into the cells at the appropriate amount, e.g., multiplicity of infection for a viral vector, according to standard transduction methods suitable for the particular target cells. Titers of virus vector to administer can vary, depending upon the target cell type and number, and the particular virus vector, and can be determined by those of skill in the art without undue experimentation. In representative embodiments, at least about 103infectious units, more preferably at least about 105infectious units are introduced to the cell.

[0125] In some embodiments, a pharmaceutical formulation is provided comprising a nucleic acid as described herein, an AAV particle, an AAV vector, or an AAV genome as described herein, in a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds also can be incorporated into the compositions. In particular embodiments, the present invention provides a pharmaceutical composition comprising a virus vector of the invention in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and will preferably be in solid or liquid particulate form.

[0126] As one of skill in the art would appreciate, a presently disclosed pharmacetucial composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such asAttorney Docket No.5470.975.WO sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Methods

[0127] In some embodiments, method of producing an AAV vector with enhanced transgene expression is provided, comprising preparing an AAV genome comprising the transgene and a mutated MAAP encoding sequence, such that MAAP expression is reduced or absent, and producing an AAV vector comprising the AAV genome, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence.

[0128] In some embodiments, a method of producing an AAV vector with modulated transgene expression is provided, comprising preparing an AAV genome comprising the transgene and a mutated MAAP encoding sequence, and producing an AAV vector comprising the AAV genome, wherein the level of transgene expression is modulated relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence. The modulation may be an increase or a decrease in transgene expression depending on whether the MAAP mutation results in a decrease or absence of MAAP expression and consequently an increase in transgene expression, an increase in MAAP expression or modified MAAP comprising, for example, an insertion in the C-terminal sequence resulting in a decrease in transgene expression, and / or an alteration in the biological activity of MAAP which may result in modified transgene expression.

[0129] In a representative embodiment, the present invention provides a method of producing an AAV vector with enhanced transgene expression, the method comprising providing to a cell in vitro, (a) a template comprising (i) a nucleic acid as described herein, and (ii) packaging signal sequences sufficient for the encapsidation of the AAV template into virus particles (e.g., one or more (e.g., two) terminal repeats, such as AAV terminal repeats), and (b) AAV sequences sufficient for replication and encapsidation of the template into viral particles (e.g., the AAV rep and AAV cap sequences encoding an AAV capsid of the invention). The template and AAV replication and capsid sequences are provided under conditions such that recombinant virus particles comprising the template packaged within the capsid are produced in the cell. The method can further comprise the step of collecting the virus particles from the cell. Virus particles may be collected from the medium and / or by lysing the cells. The cell is typically a cellAttorney Docket No.5470.975.WO that is permissive for AAV viral replication. Any suitable cell known in the art may be employed, such as mammalian cells. Also suitable are trans-complementing packaging cell lines that provide functions deleted from a replication-defective helper virus, e.g., 293 cells or other E1a trans-complementing cells.

[0130] The AAV replication and capsid sequences may be provided by any method known in the art. Current protocols typically express the AAV rep / cap genes on a single plasmid. The AAV replication and packaging sequences need not be provided together, although it may be convenient to do so. The AAV rep and / or cap sequences may be provided by any viral or non- viral vector. For example, the rep / cap sequences may be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1a or E3 regions of a deleted adenovirus vector). EBV vectors may also be employed to express the AAV cap and rep genes. One advantage of this method is that EBV vectors are episomal, yet will maintain a high copy number throughout successive cell divisions (i.e., are stably integrated into the cell as extra-chromosomal elements, designated as an EBV based nuclear episome.

[0131] As a further alternative, the rep / cap sequences may be stably carried (episomal or integrated) within a cell.

[0132] Typically, the AAV rep / cap sequences will not be flanked by the AAV packaging sequences (e.g., AAV ITRs), to prevent rescue and / or packaging of these sequences.

[0133] The template (e.g., an rAAV vector genome) can be provided to the cell using any method known in the art. For example, the template may be supplied by a non-viral (e.g., plasmid) or viral vector. In particular embodiments, the template is supplied by a herpesvirus or adenovirus vector (e.g., inserted into the E1a or E3 regions of a deleted adenovirus). As another illustration, Palombo et al., (1998) J. Virol.72:5025, describe a baculovirus vector carrying a reporter gene flanked by the AAV ITRs. EBV vectors may also be employed to deliver the template, as described above with respect to the rep / cap genes. In another representative embodiment, the template is provided by a replicating rAAV virus.

[0134] To obtain maximal virus titers, helper virus functions (e.g., adenovirus or herpesvirus) essential for a productive AAV infection are generally provided to the cell. Helper virus sequences necessary for AAV replication are known in the art. Typically, these sequences are provided by a helper adenovirus or herpesvirus vector. Alternatively, the adenovirus or herpesvirus sequences can be provided by another non-viral or viral vector, e.g., as a non-Attorney Docket No.5470.975.WO infectious adenovirus miniplasmid that carries all of the helper genes required for efficient AAV production as described by Ferrari et al., (1997) Nature Med.3:1295, and U.S. Patent Nos. 6,040,183 and 6,093,570.

[0135] Further, the helper virus functions may be provided by a packaging cell with the helper genes integrated in the chromosome or maintained as a stable extrachromosomal element. In representative embodiments, the helper virus sequences cannot be packaged into AAV virions, e.g., are not flanked by AAV ITRs.

[0136] Those skilled in the art will appreciate that it may be advantageous to provide the AAV replication and capsid sequences and the helper virus sequences (e.g., adenovirus sequences) on a single helper construct. This helper construct may be a non-viral or viral construct, but is optionally a hybrid adenovirus or hybrid herpesvirus comprising the AAV rep / cap genes.

[0137] In one particular embodiment, the AAV rep / cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector. This vector further contains the rAAV template. The AAV rep / cap sequences and / or the rAAV template may be inserted into a deleted region (e.g., the E1a or E3 regions) of the adenovirus.

[0138] In a further embodiment, the AAV rep / cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector. The rAAV template is provided as a plasmid template.

[0139] In another illustrative embodiment, the AAV rep / cap sequences and adenovirus helper sequences are provided by a single adenovirus helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained within the cell as an extrachromosomal element (e.g., as a “EBV based nuclear episome,” see Margolski, (1992) Curr. Top. Microbiol. Immun.158:67).

[0140] In a further exemplary embodiment, the AAV rep / cap sequences and adenovirus helper sequences are provided by a single adenovirus helper. The rAAV template is provided as a separate replicating viral vector. For example, the rAAV template may be provided by a rAAV particle or a second recombinant adenovirus particle.

[0141] According to the foregoing methods, the hybrid adenovirus vector typically comprises the adenovirus 5' and 3' cis sequences sufficient for adenovirus replication and packaging (i.e., the adenovirus terminal repeats and PAC sequence). The AAV rep / cap sequences and, if present, the rAAV template are embedded in the adenovirus backbone and are flanked by the 5' and 3' cisAttorney Docket No.5470.975.WO sequences, so that these sequences may be packaged into adenovirus capsids. As described above, in representative embodiments, the adenovirus helper sequences and the AAV rep / cap sequences are not flanked by the AAV packaging sequences (e.g., the AAV ITRs), so that these sequences are not packaged into the AAV virions.

[0142] Herpesvirus may also be used as a helper virus in AAV packaging methods. Hybrid herpesviruses encoding the AAV rep protein(s) may advantageously facilitate for more scalable AAV vector production schemes. A hybrid herpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep and cap genes has been described (Conway et al., (1999) Gene Therapy 6:986 and WO 00 / 17377, the disclosures of which are incorporated herein in their entireties).

[0143] As a further alternative, the virus vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and rAAV template as described by Urabe et al., (2002) Human Gene Therapy 13:1935-43.

[0144] Other methods of producing AAV use stably transformed packaging cells (see, e.g., U.S. Patent No.5,658,785).

[0145] AAV vector stocks free of contaminating helper virus may be obtained by any method known in the art. For example, AAV and helper virus may be readily differentiated based on size. AAV may also be separated away from helper virus based on affinity for a heparin substrate (Zolotukhin et al., (1999) Gene Therapy 6:973). In representative embodiments, deleted replication-defective helper viruses are used so that any contaminating helper virus is not replication competent. As a further alternative, an adenovirus helper lacking late gene expression may be employed, as only adenovirus early gene expression is required to mediate packaging of AAV virus. Adenovirus mutants defective for late gene expression are known in the art (e.g., ts100K and ts149 adenovirus mutants).

[0146] The packaging methods may be employed to produce high titer stocks of virus particles. In particular embodiments, the virus stock has a titer of at least about 105transducing units (tu) / ml, at least about 106tu / ml, at least about 107tu / ml, at least about 108tu / ml, at least about 109tu / ml, or at least about 1010tu / ml.

[0147] The disclosure is also related to methods for expressing a transgene with an AAV vector comprising an AAV genome comprising the transgene and a mutated MAAP sequence, wherein the level of transgene expression is modulated relative to an AAV vector comprising an AAVAttorney Docket No.5470.975.WO genome with a wild-type MAAP sequence. In some embodiments, a method of expressing a transgene from an AAV vector is provided, the method comprising contacting a cell with an AAV vector comprising an AAV genome comprising the transgene and a mutated MAAP sequence, such that MAAP expression is reduced or absent, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP sequence.

[0148] In some embodiments, a method of expressing a transgene from an AAV vector is provided, the method comprising contacting a cell with an AAV vector comprising an AAV genome comprising the transgene and a mutated MAAP encoding sequence, wherein the level of transgene expression is modulated relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence.

[0149] In some embodiments, the method can further comprise contacting the cell with a second AAV vector that does not comprise a mutated MAAP encoding sequence. In some embodiments, transgene expression from the second AAV vector is enhanced or modulated relative to the second AAV vector in the absence of the AAV vector comprising a mutated MAAP encoding sequence. In some embodiments, the ratio of the second AAV vector to the AAV comprising a mutated MAAP encoding sequence is between 1:10 to 10:1 on a vector genome basis, e.g., 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0150] The vectors are useful to produce a polypeptide or nucleic acid in vitro or for ex vivo gene therapy. The vectors are additionally useful in a method of delivering a nucleotide sequence to a subject in need thereof, e.g., to express a therapeutic polypeptide or nucleic acid. In this manner, the polypeptide or nucleic acid may thus be produced in vivo in the subject. The subject may be in need of the polypeptide or nucleic acid because the subject has a deficiency of the polypeptide, or because the production of the polypeptide or nucleic acid in the subject may impart some therapeutic effect, as a method of treatment or otherwise, and as explained further below.

[0151] A further aspect of the invention is a method of administering the virus vectors or capsids of the invention to subjects. In particular embodiments, the method comprises a method of delivering a nucleic acid of interest to an animal subject, the method comprising: administering an effective amount of a virus vector according to the invention to an animal subject. Administration of the virus vectors of the present invention to a human subject or an animal inAttorney Docket No.5470.975.WO need thereof can be by any means known in the art. Optionally, the virus vector is delivered in an effective dose in a pharmaceutically acceptable carrier. The enhanced transgene expression achieved by the present invention advantageously may permit lower doses of AAV vector to be administered to a subject to achieve the same result, thereby enhancing the feasibility and safety of the method.

[0152] The virus vectors of the invention can further be administered to a subject to elicit a therapeutic response. Typically, compositions of the present invention comprise an effective amount of an AAV comprising a mutated MAAP in combination with a pharmaceutically acceptable carrier. Optionally, the dosage is sufficient to produce a therapeutic response. The degree of therapeutic response conferred need not be complete or permanent.

[0153] Dosages of the virus vectors to be administered to a subject will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular virus vector, and the nucleic acid to be delivered, and can be determined in a routine manner. Exemplary doses for achieving therapeutic effects are virus titers of at least about 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015transducing units or more, e.g., about 107or 108, 109, 1010, 1011, 1012, 1013or 1014transducing units, e.g., about 1012transducing units.

[0154] In particular embodiments, more than one administration (e.g., two, three, four or more administrations) may be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, yearly, etc.

[0155] Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal, diaphragm and / or cardiac muscle], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intro-lymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragm muscle or brain). Administration can also be to a tumor (e.g., in or a near a tumor or a lymph node). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular vector that is being used.

[0156] In some embodiments, the viral vector is administered directly to the CNS, e.g., the brain or the spinal cord. Direct administration can result in high specificity of transduction of CNSAttorney Docket No.5470.975.WO cells, e.g., wherein at least 80%, 85%, 90%, 95% or more of the transduced cells are CNS cells. Any method known in the art to administer vectors directly to the CNS can be used. The vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus. The vector may also be administered to different regions of the eye such as the retina, cornea or optic nerve. The vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the vector.

[0157] The delivery vector may be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intracerebral, intraventricular, intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri- ocular (e.g., sub-Tenon's region) delivery or any combination thereof.

[0158] Typically, the viral vector will be administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired region or compartment in the CNS. In some embodiments, the vector can be delivered via a reservoir and / or pump. In other embodiments, the vector may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye or into the ear, may be by topical application of liquid droplets. As a further alternative, the vector may be administered as a solid, slow-release formulation. Controlled release of parvovirus and AAV vectors is described by international patent publication WO 01 / 91803.

[0159] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention. EXAMPLES Example 1. Mutated Membrane Associated Accessory Protein (MAAP)

[0160] AAV9 (adeno-associated virus 9) occupies a prominent position in central nervous system (CNS) gene therapy given its central role in ongoing clinical trials and an FDA approved therapeutic. AAV9 was previously shown to exhibit a capsid-promoter interaction that can alterAttorney Docket No.5470.975.WO neuronal cellular tropism in the rat and primate CNS. This example builds upon that novel finding to show that AAV9EU (AAV9 with six glutamate insertion (EU) at aa139 VP1 numbering) dramatically reduces in vivo relative transgene mRNA levels, and to some extent nuclear viral DNA. Further assessing the impact of the EU mutation on VP1, VP2, and MAAP reveals that there is only a modest reduction in relative transgene mRNA levels and no change in neuronal cellular tropism. Surprisingly, AAV9 produced with no MAAP9 exhibits an increase in relative transgene levels, and co-infusing AAV9 and AAV9 produced in the absence of MAAP9 increases in vivo transgene fluorescence intensity by 5-fold. Together, a MAAP9 related activity acts both in cis and in trans to increase AAV9 transgene mRNA levels and AAV9 transgene protein levels in vivo. These novel functions for AAV MAAP have implications that modulating MAAP could allow for fewer vectors required per cell for therapeutic effects. Results AAV9EU has reduced relative transgene mRNA levels and nuclear viral genomes

[0161] To further understand the mechanism underlying AAV9EU’s reduced transgene expression, relative transgene mRNA levels of AAV9EU and AAV9 were quantified and compared. Viruses packaged with green fluorescent protein (GFP) driven by a strong constitutive promoter, chicken beta-actin (CBA), were produced to a high titer with appropriate capsid ratios (FIG.6A). AAV9-CBA-GFP and AAV9EU-CBA-GFP were infused in equal amounts into the rat striatum. The rats were sacrificed 2 weeks post infusion, the striata harvested, RNA isolated, cDNA synthesized, and the relative transgene mRNA levels quantified. The GFP mRNA levels and endogenous GAPDH mRNA levels were measured by qPCR Taqman assays using the ΔΔct method to determine the fold change between AAV9 and AAV9EU. AAV9EU exhibits a significant decrease (p<0.05) (more than a 2000-fold reduction) in relative GFP mRNA levels compared to AAV9 (FIG.1A) which agrees with the microscopy data previously reported20.

[0162] AAV9EU induced reduction of relative GFP mRNA levels could be explained by the vector being ineffective at nuclear entry. The EU mutation is six glutamates (negatively charged) that reside in a heavily basic region of VP1 / 2 that serves as nuclear localization signals16,17. Therefore, AAV9 and AAV9EU nuclear transgene copy numbers were quantified. AAV9-CBA- GFP and AAV9EU-CBA-GFP were directly infused in equal amounts into the rat striatum. Striata were harvested 2 weeks post infusion after which nuclei were isolated, nuclear DNA purified, and GFP nuclear copies quantified with input DNA amount normalized to GAPDH using qPCRAttorney Docket No.5470.975.WO TaqMan assays. AAV9EU nuclear GFP copies were significantly reduced (p<0.05) (~10-fold less) in comparison to AAV9, suggesting that AAV9EU GFP transgenes are present within the nucleus, but in a lower amount than AAV9 (FIG. 1B). Together, these data suggest that AAV9EU is deficient at gaining nuclear entry but transcription is greatly reduced. Separate AAV9 VP1 or VP2 EU mutations do not reproduce AAV9EU oligodendrocyte cellular tropism

[0163] AAV9EU is AAV9 with 6 glutamates (EU) inserted at aa139 (VP1 numbering) resulting in the inserts in both VP1 and VP2. The individual contribution the EU mutation in VP1 and VP2 has on AAV9EU oligodendrocyte cellular tropism was investigated20. To this end, AAV9 viruses were produced with the EU mutation in only VP1 or VP2 in the AAV9 rep / cap plasmid; AAV9EU VP1 (AAVE99) or VP2 (AAV9E9) were created by mutating the start codons as previously published22but briefly, VP1 (M1L) or VP2 (M208L), respectively. A second AAV9 rep / cap plasmid with the complementary VPs was added at an equal molar amount during production. The viruses were packaged with CBA-mCherry and produced high titer virus. The viruses were infused at equal amounts into the rat striatum then harvested 2 weeks post infusion for fluorescence microscopy image analysis. AAVE99 and AAV9E9 exhibited a dominant neuronal cellular tropism with ~90% of mCherry positive cells co-localizing with a neuronal nuclear marker, NeuN (FIG. 2). Thus, AAVE99 and AAV9E9 neuronal expression proved far more robust than AAV9EU (14.7 ± 3.6%)20suggesting that EU mutations into AAV9 VP1 and VP2 individually cannot account for the dominant oligodendrocyte cellular tropism of AAV9EU. AAV9 VP1 or VP2 EU mutations in combination with MAAP9 or MAAP9EU do not recapitulate AAV9EU oligodendrocyte cellular tropism

[0164] The AAV9EU vector does not support robust transgene expression and transduces mostly oligodendrocytes compared to AAV9, AAVE99, and AAV9E9 (published data20and FIG. 2) suggesting additional factor(s) contribute to the AAV9EU phenotype. The EU mutation into AAV9 cap at aa139 results in a RRKRRK insertion into the C-terminus of MAAP9 resulting in MAAP9EU (FIG.7). Although MAAP is not known to have a role outside of vector production, it was assessed if MAAP9EU alone could alter AAV9 cellular tropism.

[0165] AAV9 / MAAP9EU-CBA-GFP was produced by mutating the MAAP9 start site (CTG to CCG) while preserving VP1 / VP2 protein sequence in the rep / cap plasmid9-11. A MAAP9EU rep / cap plasmid that expressed only MAAP9EU and no VPs was cloned by mutating the VPs startAttorney Docket No.5470.975.WO sites. The two rep / cap plasmids were combined in a 1:2 molar ratio (MAAP: VPs) for virus production, the virus was harvested after 48 hours from only the cell pellet, and high titer was produced. AAV9 / MAAP9EU-CBA-GFP and AAV9-CBA-mCherry of equal amounts were mixed 1:1 and infused into the rat striatum to compare reporter gene expression pattern 2 weeks post infusion. Both viruses exhibited robust transgene expression with a high degree of co-localization in cells morphologically resembling neurons (FIG. 3A). Therefore, MAAP9EU alone does not appear to contribute to the oligodendrocyte cellular tropism of AAV9EU.

[0166] Thus, it was assessed if either the AAV9 VP1 or VP2 EU mutation in combination with MAAP9EU reduces neuronal cellular tropism. AAV9 with the EU mutation in VP1 (AAVE99) or VP2 (AAV9E9) were produced with either MAAP9 or MAAP9EU then directly compared to AAV9 for differences in cellular tropism. VP1, VP2, and MAAP mutants in AAV9EU and AAV9 were cloned as described above with the relevant MAAP start site mutated that preserved VP1 / VP2 protein sequence9-11. All viruses (AAVE99 / MAAP9-CBA-GFP, AAV9E9 / MAAP9-CBA-GFP, AAVE99 / MAAP9EU-CBA-GFP, AAV9E9 / MAAP9EU-CBA-GFP) were produced as previously described using two rep / cap plasmids at equal molar amounts for all components22, harvested after 48 hours from only the cell pellet, and high titer virus produced. The mutant viruses were mixed 1:1 with AAV9-CBA-mCherry in equal amounts and infused into the rat striatum to compare cellular tropism at 2 weeks post infusion by confocal fluorescence microscopy analysis. All mutant viruses exhibited robust transgene expression in cells morphologically appearing to be neurons, with substantial co-localization with AAV9-CBA-mCherry expression (FIGS. 3B-3C). Importantly, no combination of AAV9EU mutation into VP location or MAAP9 / MAAP9EU resulted in the oligodendrocyte cellular tropism seen with AAV9EU20. These results suggest that the EU mutation in VP1 or VP2 in combination with MAAP9EU is not responsible for AAV9EU oligodendrocyte cellular tropism. MAAP9 mutants alter relative transgene mRNA levels in vivo

[0167] Given the 2000-fold difference in relative transgene mRNA levels between AAV9 and AAV9EU the relative transgene mRNA levels in the single AAV9 VP1 EU, VP2 EU, and MAAP9 mutants were measured. To quantify the relative transgene mRNA levels, AAV9-CBA-GFP, AAV9 / no MAAP-CBA-GFP, AAV9 / MAAP9EU-CBA-GFP, AAVE99 / MAAP9-CBA-GFP, AAV9E9 / MAAP9-CBA-GFP, AAVE99 / MAAP9EU-CBA-GFP, and AAV9E9 / MAAP9EU- CBA-GFP were produced, harvested after 48 hours from only the cell pellet, and purified to a highAttorney Docket No.5470.975.WO titer. Each virus was directly infused in equal amounts into the rat striatum, and the striata harvested at 2 weeks post infusion. The RNA was processed and analyzed as described above. Surprisingly, AAV9 / no MAAP-CBA-GFP exhibited a significant increase (p>0.05) in fold change in relative GFP mRNA levels compared to AAV9 (FIG.4). Upon repeating the experiment, viral nuclear DNA copies were assessed and exhibited similar copy numbers, so nuclear viral copies did not explain the difference in relative GFP mRNA levels. The EU mutation in AAV9 VP1, VP2, or MAAP9 reduces the fold change in relative GFP mRNA levels compared to AAV9 (FIG. 4). Together, the EU mutation in VP1 or VP2 in combination with MAAP9 does not replicate the transgene mRNA reduction of AAV9EU. Further, these findings suggest that MAAP9EU and no MAAP have different distinct in vivo actions. A MAAP9 related activity can modulate AAV9 in vivo transgene expression in cis and in trans

[0168] Given that AAV9 / no MAAP resulted in a substantial increase in the relative GFP mRNA levels (FIG.4) the potential role of MAAP9 outside of production was further assessed by directly comparing it to AAV9 in vivo. AAV9 / no MAAP-CBA-GFP virus was created as described above, harvested after 48 hours from only the cell pellet, and high titer virus was produced with appropriate VP ratios (FIG.6B). AAV9 / no MAAP-CBA-GFP and AAV9-CBA-mCherry at equal amounts were mixed 1:1 and infused into the rat striatum then at 2 weeks post infusion the cellular tropism was compared using confocal fluorescence microscopy. There was no discernible difference in transduced cell morphology (neuronal) between AAV9 and the AAV9 / no MAAP virus (FIG.3D).

[0169] However, it was noticed that the microscopy exposure settings in experiments involving mixing AAV9 / no MAAP and AAV9 viruses needed to be substanially adjusted, a result that is not explained by titer or infused virus amount. This preliminary observation suggested that mixing AAV9 with AAV9 / no MAAP increases transgene protein levels from both vectors in vivo. To visualize this observation, AAV9-CBA-mCherry was mixed 1:1 at equal amounts with either AAV9-CBA-GFP or AAV9 / no MAAP-CBA-GFP and infused bilaterally into the striata of 6 rats. After 2 weeks the animals were sacrificed, perfused, fixed, and the brains sliced in 40µm sections sequentially through the striatum. Brightfield and native flourescence images (4X) were taken through the infusion site to identify the section where the injector pierces the corpus callosum to make analogous comparisons between the virus mixes. Image analysis was performed on 11Attorney Docket No.5470.975.WO sequential sections including and after the corpus callosum break (FIG.8). The area above a set threshold of native fluorescence intensity within the striatum was used in analysis for intra-animal comparisions. Higher transgene protein amounts (average 5-fold more) occurs when AAV9-CBA- mCherry and AAV / no MAAP-CBA-GFP are mixed compared to the mix of viruses produced with MAAP9 (Table 2). The slide scanner analysis was confirmed by using confocal flourecence microscopy image analysis of the first and last sections to determine and compare flourescence intensity density (FIG.9). These results suggest that mixing AAV9 / no MAAP with AAV9 results in an in trans effect on transgene protein levels. Table 2. When mixed, AAV9 / no MAAP and AAV9 exhibit enhanced fluorescence intensity in the rat striatum AAV9-CBA-mCherry AAV9-CBA-mCherry Fold Difference + AAV9-CBA-GFP + AAV9 / no MAAP-CBA-GFP (MAAP vs. no

[0170] AAV9-CBA-mCherry was mixed 1:1 and co-infused with either AAV9-CBA-GFP or AAV9 / no MAAP-CBA-GFP into 6 rat striata. All viruses were of equal titer and an equal volume was infused bilaterally into the rat striatum for intra-animal comparisons. The average area (µm2) ± SEM of native fluorescence signal intensity above a threshold (20,000) in eleven 40µm sections of comparable infusion site sections within the rat striatum. The brain section where the injector tract transects the corpus callosum is used as the initial section of the infusion site. Images were collected on a slide scanner using the same settings. *a,bAll intra-animal paired comparisons are statistically significant using Wilcoxon test, P<0.05.

[0171] While the data from all animals showed the same trend, the nuclear viral genome copies and relative transgene mRNA levels between the experimental conditions was also determined. Viruses were mixed and infused as above. After 2 weeks, nuclear DNA and RNA were isolated from each striatium then processed and analyzed as above. Comparable nuclear vector copies of GFP and mCherry were obtained from both comparisons suggesting that the difference inAttorney Docket No.5470.975.WO transgene protein levels is not due to differences in nuclear entry or transduction (FIG. 10A). Further, the relative levels of both GFP and mCherry mRNA compared to GAPDH were similar in both conditions (FIG. 10B). These results suggest that MAAP9 exhibits an activity that acts both in cis and in trans to modulate viral transgene protein levels in vivo. This activity is independent of altering nuclear viral copies or transgene mRNA levels. Further, MAAP9 has a role outside of AAV production and association with the cellular membrane and exosomes. Discussion

[0172] Over the years, significant efforts have focused upon manipulating AAV capsid structure to create new AAVs that exhibit novel, clinically beneficial properties. The majority of AAV9 based studies have utilized either 7 or 21 amino acid insertions between AAV9 aa 588 and 589, part of the VR hypervariable region which displays the peptides on the surface of the capsid23,24,25. Recently Powell et al. showed that a six glutamate insertion (EU mutation) into AAV9 VP1 / 2 at aa139 resulted in dramatic alterations in in vivo transgene expression, leading to the novel concept of a heretofore unknown AAV9 capsid / promoter interaction20. In order to further define the nature of this AAV9 capsid / promoter interaction the present studies investigated the singular influence of the EU mutation on VP1, VP2, or MAAP.

[0173] The present investigations found that the EU mutation into AAV9 VP1 alone or VP2 alone did influence the relative mRNA levels in the rat striatum but did not recapitulate the dramatic reduction in transgene expression of AAV9EU. In contrast, the EU mutation into both VP1 and VP2 (AAV9EU) resulted in a 2000-fold reduction in in vivo relative transgene mRNA levels and a 10-fold reduction in nuclear transgene DNA (FIGS.1A-1B). In addition, AAV9EU contains a mutant MAAP that contributes to the reduction in transduction given that MAAP9EU alone exhibited a reduction in relative mRNA levels (FIG. 4). Although both DNA and mRNA levels could contribute to the reduction in transduction in AAV9EU, the mechanisms are unclear. Recent studies have shown that the capsid can alter the epigenetic state of its genome26,27. Further, in vitro studies revealed that protein levels and mRNA levels are not necessarily correlated19. While ours and other’s observations were in experiments using different AAV serotypes and tissues, together, they all support that an AAV VP1 / 2 exhibits activity to promote transduction outside of lysosome escape and nuclear localization. The activities and contribution of VP1, VP2, and MAAP to AAV9 transduction are still unclear. However, it is striking that when EU mutations into VP1 or VP2 were combined with MAAP9EU, the cellular tropism (FIGS.3A-3D) and relative mRNA levelsAttorney Docket No.5470.975.WO (FIG.4) were similar. These results suggest that VP1 or VP2 can compensate for the EU mutation in the other.

[0174] As importantly, further investigations of other potential factors that underly the AAV9EU phenotype led to the discovery of MAAP9 influence on in vivo transgene expression. Following the initial discovery of the MAAP sequence in the AAV capsid coding sequence, subsequent studies established that MAAP facilitated the movement of AAV particles into exosomes, an event that resulted in recombinant virus secretion into the media during virus production9-11. Having the sequence and location of MAAP, it was noted that the EU mutation into AAV9 VP1 and VP2 resulted in a RRKRRK insertion into the C-terminus of MAAP9 (MAAP9EU). Therefore, these studies had inserted basic amino acids in an already highly basic region (potentially NLS signals) and into the proposed membrane binding domain of MAAPs. MAAP2 truncations immediately before these insertions were previously shown to affect titer and capsid protein levels11, but C- terminal tags FLAG and GFP did allow for several serotypes of MAAP to localize to membranes9,10. Together, how the EU mutation altered MAAP9 function remains an outstanding question, but the absence of MAAP9 increased relative transgene mRNA levels in the rat striatum (FIG.4).

[0175] In the earlier VP1 and VP2 mutation studies (FIG. 2), viruses were produced with both MAAP9 and MAAP9EU on the respective rep / cap plasmids. However, it is unknown if mutations to the VP1 start site or VP2 start site altered the expression levels of MAAP9 and MAAP9EU during production. Studies from the Church lab found that when a MAAP and no MAAP AAV2 virus were produced together, the MAAP virus out competes the no MAAP virus in terms of titer9. Although the studies involved different capsid and MAAP configurations, and without being bound by theory, it is unlikely that one MAAP configuration out competes another given similar virus titers across different manipulations. Also, the results show that the different configurations of glutamate inserts and MAAPs exhibit a similar cellular tropism (neuronal) (FIG. 2) as the vectors produced with only MAAP9 or MAAP9EU then mixed prior to infusion (FIGS.3A-3D). While the experiments are unable to discern the amounts of each MAAP during production and how that translates to in vivo outcomes; the in vivo neuronal cellular tropism is independent of MAAP9 and MAAP9EU during production (FIG. 2 and FIGS. 3A-3D). AAV2 and MAAP2 in vitro studies indicate that mutations in MAAP2 can alter the titer and the authors further suggest that levels of MAAP2, AAP, and VPs are required in a certain ratio11. While these results suggestAttorney Docket No.5470.975.WO that mutations in MAAP or altered molar amount effect titers, purified AAV9 / no MAAP and AAV9 have similar titers and VP ratios (FIGS.6A-6B).

[0176] The studies removing MAAP9 from production then assessing the in vivo outcome suggests that MAAP has a role in modulating transgene levels in vivo. The absence of MAAP9 during production lead to an increase in relative GFP mRNA levels in vivo (FIG. 4). Previous studies only assessed the lack of MAAP during production, which results in more Rep and Cap protein levels11, an increase in aberrant AAV genome packaging11, and a reduced titer when produced together with a MAAP containing virus9. The studies did not assess the packaged genomes, but certainly it is necessary to further understand in vivo outcomes. Packaging of aberrant genomic material is possible, and it is highly undesirable in clinical grade vectors28. A possible link between MAAP and AAV packaging fidelity would certainly impact large scale or AAV library production.

[0177] The studies show that MAAP9 can modulate viral transgene protein levels in vivo. When AAV9 was mixed with AAV9 / no MAAP a significant increase in transgene protein levels were seen for both vectors (Table 2). This in vivo interaction establishes a novel function for AAV9 MAAP distinct from the currently accepted dogma that some AAV MAAPs facilitate viral exosome trafficking into the production media10,12. Given that previous studies have shown that MAAPs of different serotypes exhibit a variable ability to traffic the virus into the media10, certainly different serotype MAAPs could have distinctly different roles. It should be noted that in the experiments the recombinant AAV9 virus was purified after 48 hours from the cell pellet, not the culture media, and MAAP9 exhibits minimal ability to traffic AAV9 to the culture media10. Thus, exosome association would not be expected to influence the virus preps or the experimental outcomes. The results suggests that AAV9 / no MAAP alone increases relative transgene expression (FIG.4); however, when the same virus is mixed with AAV9 produced with MAAP there is no discernable difference in relative transgene mRNA levels (FIG. 10B) but an increase in both transgene protein levels (Table 2). The mixing experiments do suggest caution be used when mixing AAV / MAAP serotypes or assessing an AAV library where multiple variants could exist within a cell.

[0178] MAAP trafficking AAVs into production media suggests that MAAP and VPs interact. An interaction of MAAP and VPs during production is certainly reasonable and attractive, yet studies looking for binding evidence proved unsuccessful10. Studies using in vitro localization have shownAttorney Docket No.5470.975.WO that AAV2 capsid proteins remain in the cytoplasm with a MAAP2 mutant which suggests a potential binding interaction between MAAP and VPs11. While MAAP activity during production has a more straight-forward explanation, an in vivo mechanism is not straight-forward. It is possible that MAAP is part of the purified vector or is facilitating capsid manipulation during production in such a way to influence transgene protein expression on a cellular level. Work from this lab and others have highlighted how the AAV capsid can modify in vivo transgene expression20and that a capsid / subunits can direct the epigenetic state of the transgene that determines its ability to express26,27. Considering AAV’s unique life cycle, MAAP could be an integral component in AAV latency. The current studies show that no MAAP increases protein expression, suggesting MAAP9 functions to lower protein expression in vivo potentially tailoring genome expression for latency. It has been shown that the AAV2 ITRs are able to support a low level of expression in absence of a promoter29, so MAAP could act to further control AAV genome expression. MAAP comprises a novel factor that can be manipulated to alter AAV expression that does not require space on the transgene. Methods Plasmid Cloning

[0179] The CBA-mCherry construct was previously described20with the CBA-GFP version differing in only the reporter gene. GFP was inserted into the CBA-mCherry backbone using EcoRI and HindIII restriction sites.

[0180] To create hybrid VP viruses the respective start codons were mutated as published18,22. Briefly, the VP1 start site was mutated to M1L, the VP2 start site was mutated to T138A, and the VP3 start site mutated to M208L. Mutations were introduced using mutagenic PCR primers (Table 3) (IDT, Coralville, IA) with two rounds of PCR using PfuUltra II Hotstart PCR Master Mix (Agilent, Santa Clara, CA 600850), the appropriate sized PCR products were then assembled into SalI-BsiWI sites in rep / cap plasmid using NEBuilder HiFi DNA assembly master mix (NEB, Ipswich, MA. cat no:E2621S).

[0181] Cloning of MAAP9EU helper plasmid was synthesized (IDT, Coralville, IA) with mutated VP1 and VP2 start sites (did not alter the VP protein sequence), a stop codon before the MAAP start site, a stop site after MAAP, and VP3 was truncated at aa156 (VP1). The synthesized DNA was cloned into rep / cap plasmid backbone at SwaI-XbaI sites using NEBuilder HiFi DNA assembly master mix (NEB, Ipswich, MA. cat no:E2621S).Attorney Docket No.5470.975.WO Nuclear DNA isolation and qPCR analysis

[0182] Nuclei were isolated from 50mg of resected rat striata tissue using NE-PER reagent kit (Thermo-Fisher, Waltham, MA. cat no: 78833) with modifications. After nuclei were obtained, they were gently washed three times with ice-cold 1XPBS (phosphate- buffered saline pH 7.4) and centrifuged for 1 min at 16000xg. The supernatant was removed from the pelleted nuclei then DNA extracted using DNeasy blood and tissue kit (Qiagen, Hilden, Germany cat no: 69504) with RNaseA treatment as per the protocol. The nuclear DNA was eluted with molecular biology grade water (Corning, Manassas, VA cat no: 46-000-CM) for use in qPCR.10 ng of input nuclear DNA was used in each reaction. Taqman expression assay (Thermo-Fisher, Waltham, MA.) for endogenous control GAPDH (rn01775763_g1) and transgenes GFP (mr04329676_mr) and mCherry (mr07319438_mr) using TaqMan fast advanced mastermix (Thermo-Fisher, Waltham, MA. cat no: 4444556) per instructions on Quantstudio3 (AppliedBiosystems, Waltham, MA.) Transgene standard curve was made using linearized double stranded transgene from plasmid then diluted from 1.0E10 copies-1.0E2 copies in uninjected striatum nuclear DNA. Uninjected nuclear DNA was run in standard curve to determine GAPDH values to calculate genome ng amount in each sample. Each sample was run in triplicate then averaged. The transgene copies and amount of total DNA were calculated using the appropriate standard curve run in parallel. qPCR data was exported and tabulated in Excel (Microsoft, Redmond, WA). All data statistical and graphing was completed in Prism (Irvine, CA). qPCR data significance was determined by Mann-Whitney test in Prism (Irvine, CA). RNA isolation and qPCR analysis

[0183] RNA was isolated from 40-50 mg resected striata tissue using Qiagen RNeasy lipid kit (Qiagen, Hilden, Germany cat no:74804) with on-column DNaseI treatment as per the manual. RNA was eluted in RNase free water. 2.5µg of RNA was used for cDNA synthesis with Superscript IV VILO master mix with ezDNase enzyme (Thermo-Fisher, Waltham, MA. cat no: 11766050). cDNA was then used in qPCR Taqman Expression assays (Thermo-Fisher, Waltham, MA.) for control GAPDH (rn01775763_g1) and transgenes GFP (mr04329676_mr) and mCherry (mr07319438_mr) using TaqMan fast advanced mastermix (Thermo-Fisher, Waltham, MA. cat no: 4444556) per instructions on Quantstudio3 (AppliedBiosystems, Waltham, MA.). Each sample was run in triplicate and repeated at least twice. The relative GFP or mCherry mRNA levels were related to GAPDH then compared to the control within the same animal to determine fold change.Attorney Docket No.5470.975.WO qPCR data was exported and tabulated in Excel (Microsoft, Redmond, WA). All data statistical and graphing was completed in Prism (Irvine, CA). qPCR data significance was determined by Mann-Whitney test in Prism (Irvine, CA). Virus Production

[0184] All transgenes used the same Chicken Beta-actin promoter (CBA) with either GFP or mCherry as fluorescent reporters. The virus was produced in HEK293 cells as previously described16. Briefly, polyethylenimine max (PEI) was used for the triple transfection of the rep / cap plasmid(s), the pXX6-80 helper plasmid, and the transgene CBA-GFP-hGHpolyA or CBA- mcherry-hGHpolyA with AAV2 inverted terminal repeats. For chimera / hybrid capsids the 2 rep / cap plasmid were used at equal molar amounts. Cells were harvested 48 hr post-transfection and purified as previously described20with triple phase partitioning30followed by cesium chloride ultracentrifugation. The relative capsid subunit ratio was assessed by running boiled samples in a final 1X concentration of NovexTMTris-Glycine SDS Sample buffer (Invitrogen, Waltham, MA. cat no: LC2676) and 1X NuPAGETMsample reducing agent (Invitrogen, Waltham, MA. cat no: NP0004). Samples loaded onto NovexTM4-12% Tris-Glycine gel (Invitrogen, Waltham, MA. cat no: XP4120) with PageRuler protein weight marker (Invitrogen, Waltham, MA. cat no: 25616). The gel was run at 100V for 2 hours in Tris-Glycine SDS Running Buffer (Invitrogen, Waltham, MA. cat no: LC2675) then stained with PageBlue Protein Staining Solution (Invitrogen, Waltham, MA. cat no: 24620) per the manufacturer’s protocol. The gels were imaged on an AmershamTMImager 600 (Cytiva, Marlborough, MA). Animals and Stereotactic Infusions

[0185] All of the animals were male and female Sprague-Dawley rats (Charles River Laboratories) weighing between 175 and 250 grams at the time of intracranial injection. The animals were maintained on a 12-hr light-dark cycle and had free access to water and food. For all animal studies, care and procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all procedures received prior approval by the University of North Carolina Institutional Animal Care and Usage Committee.

[0186] For the virus vector infusions, first the animals were anesthetized with inhalational isoflurane (3% induction, 2.5 % maintenance) and placed into a stereotactic frame. Using a 32G stainless steel injector and a infusion pump, animals received 1 μL of each vector (1.0E9vg / µL) into the striatum over 5 minutes either unilaterally or bilaterally (0.5-1.0 mm anterior to bregma,Attorney Docket No.5470.975.WO 3.0 mm lateral, and 5.5 mm vertical, according to the atlas of Paxinos and Watson31. The injector was left in place for 1 min post-infusion in order to allow diffusion from the injector. Fluorescence visualization and analysis

[0187] Two weeks after AAV vector infusion, animals were euthanized for confocal microscopy evaluation or tissue collection using the drop isoflurane method. For the confocal microscopy the animals were perfused transcardially with ice-cold 100 mM PBS (pH 7.4), followed by 4% paraformaldehyde in PB (pH 7.4). After brains were post-fixed 12–48 hr at 4°C in the paraformaldehyde-PB, 40-μm coronal sections were cut using a vibrating blade microtome for confocal or slide scanning determination of mCherry or GFP fluorescence. For NeuN detection, NeuN primary antibody (Millipore, Bedford, MA, cat no: MAB377) at 1:500 followed by Secondary goat anti-mouse Alexa 488 (Invitrogen Waltham, MA. Cat no: A11032). For the DNA and mRNA determinations, fresh striatal tissue was dissected immediately following euthanasia.

[0188] For native fluorescence visualization sections were mounted, and native fluorescence was visualized using an Olympus FV3000RS confocal microscope in the UNC Neuroscience Center Confocal and Multiphoton Imaging Core. Images were taken using 20X objective and displayed as maximum projections. For confocal imaging fluorescence density IMARIS software (Oxford Instruments, UK) was used.

[0189] For slide scanner analysis, sections were sequentially mounted and visualized with brightfield and fluorescence at 4X on the Evident / Olympus VS200 (Waltham, MA.) at the UNC Hooker Imaging Core. A total of 6 animals were imaged through the entire infusion site. The average area of native fluorescence intensity signal above 20,000 was determined for 11 sequential sections for each condition using QuPath32. The fold difference was determined by averaging the values from 11 sections followed by intra-animal comparisons. Statistical analysis performed in Prism (Irvine, CA) using Wilcoxon test. Table 3: DNA primers used for cloning plasmids within Example 1. Primers used for cloning all constructs used in the experiments. All were made and obtained from IDT (Coralville, IA). Primer Name Sequence Use l ’ iAttorney Docket No.5470.975.WO R 5’ ggagccgccttagcc (SEQ ID NO: 7) 9EUT138A F’ 5’ ggctaaggcggaggag (SEQ ID NO: 8) T138A (VP2) mutation primers for R 5’ ctcctccgccttagcc (SEQ ID NO: 9) AAV9EU 9 g

[0190] References 1. Kerr, J.R. (2006). Parvoviruses (Hodder Arnold ;Distributed in the United States of America by Oxford University Press). 2. Wu, Z., Asokan, A., and Samulski, R.J. (2006). Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther 14, 316-327.10.1016 / j.ymthe.2006.05.009. 3. Kuzmin, D.A., Shutova, M.V., Johnston, N.R., Smith, O.P., Fedorin, V.V., Kukushkin, Y.S., van der Loo, J.C.M., and Johnstone, E.C. (2021). The clinical landscape for AAV gene therapies. Nat Rev Drug Discov 20, 173-174.10.1038 / d41573-021-00017-7. 4. Rose, J.A., Maizel, J.V., Jr., Inman, J.K., and Shatkin, A.J. (1971). Structural proteins of adenovirus-associated viruses. J Virol 8, 766-770.10.1128 / JVI.8.5.766-770.1971. 5. Buller, R.M., and Rose, J.A. (1978). Characterization of adenovirus-associated virus- induced polypeptides in KB cells. J Virol 25, 331-338.10.1128 / JVI.25.1.331-338.1978. 6. Sonntag, F., Schmidt, K., and Kleinschmidt, J.A. (2010). A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci U S A 107, 10220- 10225.10.1073 / pnas.1001673107. 7. Sonntag, F., Kother, K., Schmidt, K., Weghofer, M., Raupp, C., Nieto, K., Kuck, A., Gerlach, B., Bottcher, B., Muller, O.J., et al. (2011). The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes. J Virol 85, 12686-12697.10.1128 / JVI.05359-11. 8. Earley, L.F., Powers, J.M., Adachi, K., Baumgart, J.T., Meyer, N.L., Xie, Q., Chapman, M.S., and Nakai, H. (2017). Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11. J Virol 91.10.1128 / JVI.01980-16. 9. Ogden, P.J., Kelsic, E.D., Sinai, S., and Church, G.M. (2019). Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design. Science 366, 1139-1143.10.1126 / science.aaw2900. 10. Elmore, Z.C., Patrick Havlik, L., Oh, D.K., Anderson, L., Daaboul, G., Devlin, G.W., Vincent, H.A., and Asokan, A. (2021). The membrane associated accessory protein is an adeno-associated viral egress factor. Nat Commun 12, 6239.10.1038 / s41467-021-26485- 4.Attorney Docket No.5470.975.WO 11. Galibert, L., Hyvonen, A., Eriksson, R.A.E., Mattola, S., Aho, V., Salminen, S., Albers, J.D., Peltola, S.K., Weman, S., Nieminen, T., et al. (2021). Functional roles of the membrane-associated AAV protein MAAP. Sci Rep 11, 21698.10.1038 / s41598-021- 01220-7. 12. Cheng, M., Dietz, L., Gong, Y., Eichler, F., Nammour, J., Ng, C., Grimm, D., and Maguire, C.A. (2021). Neutralizing Antibody Evasion and Transduction with Purified Extracellular Vesicle-Enveloped Adeno-Associated Virus Vectors. Hum Gene Ther 32, 1457-1470.10.1089 / hum.2021.122. 13. Pillay, S., Meyer, N.L., Puschnik, A.S., Davulcu, O., Diep, J., Ishikawa, Y., Jae, L.T., Wosen, J.E., Nagamine, C.M., Chapman, M.S., and Carette, J.E. (2016). An essential receptor for adeno-associated virus infection. Nature 530, 108-112.10.1038 / nature16465. 14. Dudek, A.M., Zabaleta, N., Zinn, E., Pillay, S., Zengel, J., Porter, C., Franceschini, J.S., Estelien, R., Carette, J.E., Zhou, G.L., and Vandenberghe, L.H. (2020). GPR108 Is a Highly Conserved AAV Entry Factor. Mol Ther 28, 367-381. 10.1016 / j.ymthe.2019.11.005. 15. Girod, A., Wobus, C.E., Zadori, Z., Ried, M., Leike, K., Tijssen, P., Kleinschmidt, J.A., and Hallek, M. (2002). The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity. J Gen Virol 83, 973- 978.10.1099 / 0022-1317-83-5-973. 16. Grieger, J.C., Snowdy, S., and Samulski, R.J. (2006). Separate basic region motifs within the adeno-associated virus capsid proteins are essential for infectivity and assembly. J Virol 80, 5199-5210.10.1128 / JVI.02723-05. 17. Hoque, M., Ishizu, K., Matsumoto, A., Han, S.I., Arisaka, F., Takayama, M., Suzuki, K., Kato, K., Kanda, T., Watanabe, H., and Handa, H. (1999). Nuclear transport of the major capsid protein is essential for adeno-associated virus capsid formation. J Virol 73, 7912- 7915.10.1128 / JVI.73.9.7912-7915.1999. 18. Johnson, J.S., Li, C., DiPrimio, N., Weinberg, M.S., McCown, T.J., and Samulski, R.J. (2010). Mutagenesis of adeno-associated virus type 2 capsid protein VP1 uncovers new roles for basic amino acids in trafficking and cell-specific transduction. J Virol 84, 8888- 8902.10.1128 / JVI.00687-10. 19. Robinson, T.M., Ho, M.L., Wahlig, B., Gough, V., Banta, A., Reyes Gamas, K., Kang, B., Lee, E., Chen, W., and Suh, J. (2020). An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription. Virology 546, 127-132.10.1016 / j.virol.2020.04.008. 20. Powell, S.K., Samulski, R.J., and McCown, T.J. (2020). AAV Capsid-Promoter Interactions Determine CNS Cell-Selective Gene Expression In Vivo. Mol Ther 28, 1373- 1380.10.1016 / j.ymthe.2020.03.007. 21. Bohlen, M.O., McCown, T.J., Powell, S.K., El-Nahal, H.G., Daw, T., Basso, M.A., Sommer, M.A., and Samulski, R.J. (2020). Adeno-Associated Virus Capsid-Promoter Interactions in the Brain Translate from Rat to the Nonhuman Primate. Hum Gene Ther 31, 1155-1168.10.1089 / hum.2020.196. 22. Warrington, K.H., Jr., Gorbatyuk, O.S., Harrison, J.K., Opie, S.R., Zolotukhin, S., and Muzyczka, N. (2004). Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J Virol 78, 6595-6609. 10.1128 / JVI.78.12.6595-6609.2004.Attorney Docket No.5470.975.WO 23. Deverman, B.E., Pravdo, P.L., Simpson, B.P., Kumar, S.R., Chan, K.Y., Banerjee, A., Wu, W.L., Yang, B., Huber, N., Pasca, S.P., and Gradinaru, V. (2016). Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204-209.10.1038 / nbt.3440. 24. Hanlon, K.S., Meltzer, J.C., Buzhdygan, T., Cheng, M.J., Sena-Esteves, M., Bennett, R.E., Sullivan, T.P., Razmpour, R., Gong, Y., Ng, C., et al. (2019). Selection of an Efficient AAV Vector for Robust CNS Transgene Expression. Mol Ther Methods Clin Dev 15, 320-332.10.1016 / j.omtm.2019.10.007. 25. Stanton, A.C., Lagerborg, K.A., Tellez, L., Krunnfusz, A., King, E.M., Ye, S., Solomon, I.H., Tabebordbar, M., and Sabeti, P.C. (2023). Systemic administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque CNS. Med 4, 31-50 e38.10.1016 / j.medj.2022.11.002. 26. Gonzalez-Sandoval, A., Pekrun, K., Tsuji, S., Zhang, F., Hung, K.L., Chang, H.Y., and Kay, M.A. (2023). The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner. Nat Commun 14, 2448. 10.1038 / s41467-023-38106-3. 27. Loeb, E.J., Havlik, P.L., Elmore, Z.C., Rosales, A., Fergione, S.M., Gonzalez, T.J., Smith, T.J., Benkert, A.R., Fiflis, D.N., and Asokan, A. (2024). Capsid-mediated control of adeno-associated viral transcription determines host range. Cell Rep 43, 113902. 10.1016 / j.celrep.2024.113902. 28. Penaud-Budloo, M., Francois, A., Clement, N., and Ayuso, E. (2018). Pharmacology of Recombinant Adeno-associated Virus Production. Mol Ther Methods Clin Dev 8, 166- 180.10.1016 / j.omtm.2018.01.002. 29. Haberman, R.P., McCown, T.J., and Samulski, R.J. (2000). Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A / D junction element. J Virol 74, 8732-8739.10.1128 / jvi.74.18.8732-8739.2000. 30. Yu, Z., Zhou, S., Luo, N., Ho, C.Y., Chen, M., and Chen, H. (2020). TPP Combined with DGUC as an Economic and Universal Process for Large-Scale Purification of AAV Vectors. Mol Ther Methods Clin Dev 17, 34-48.10.1016 / j.omtm.2019.11.009. 31. Paxinos, G., and Watson, C. (1998). The rat brain in stereotaxic coordinates, 4th Edition (Academic Press). 32. Bankhead, P., Loughrey, M.B., Fernandez, J.A., Dombrowski, Y., McArt, D.G., Dunne, P.D., McQuaid, S., Gray, R.T., Murray, L.J., Coleman, H.G., et al. (2017). QuPath: Open source software for digital pathology image analysis. Sci Rep 7, 16878.10.1038 / s41598- 017-17204-5. Example 2. MAAP9 Expression Modulates In Vivo Transduction

[0191] To track MAAP9 expression in vivo, it was fused with mCherry. mCherry-MAAP9 overexpression led to a decrease in mCherry expression in vivo along with differences in cellular localization, together suggesting that MAAP9 expression works to modulate in vivo transduction, as described below.

[0192] An mCherry-MAAP9 fusion driven by a CBA promoter (Chicken β-actin) was packaged in AAV9 (AAV9-CBA-mCherry-MAAP9-3xFlag). Importantly, the CBA promoter allows forAttorney Docket No.5470.975.WO high expression during vector production and in vivo. Both AAV9-CBA-mCherry-MAAP9- 3xFlag and AAV9-CBA-mCherry were infused in an equal amount into the rat striatum. Native mCherry expression was assessed 2 weeks post-infusion. Imaging results in FIG.11A suggest that overexpression of mCherry-MAAP9 during vector production, in vivo, or both results in a substantial decrease in transgene expression in vivo. Thus, MAAP9 overexpression has detrimental effects on AAV in vivo transduction. FIG.11B indicates that mCherry-MAAP9 fusion expression exhibits cellular morphology consistent with membrane localization suggesting that it can function in vivo as it has been described in vitro. In addition, mCherry- MAAP9 fusion expression results in lower cellular transduction further supporting a novel role for MAAP9 in vivo transduction. The results do not distinguish whether the phenotype is due to MAAP9 overexpression during production, in vivo, or both.

[0193] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

Attorney Docket No.5470.975.WO CLAIMS What is claimed is:

1. A method of producing an adeno-associated virus (AAV) vector with enhanced transgene expression, comprising preparing an AAV genome comprising the transgene and a mutated membrane associated accessory protein (MAAP) encoding sequence, such that MAAP expression is reduced or absent, and producing an AAV vector comprising the AAV genome, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence.

2. A method of producing an AAV vector with modulated transgene expression, comprising preparing an AAV genome comprising the transgene and a mutated MAAP encoding sequence, and producing an AAV vector comprising the AAV genome, wherein the level of transgene expression is modulated relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence.

3. A method of expressing a transgene from an AAV vector, the method comprising contacting a cell with an AAV vector comprising an AAV genome comprising the transgene and a mutated MAAP encoding sequence, such that MAAP expression is reduced or absent, wherein the level of transgene expression is enhanced relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence.

4. A method of expressing a transgene from an AAV vector, the method comprising contacting a cell with an AAV vector comprising an AAV genome comprising the transgene and a mutated MAAP encoding sequence, wherein the level of transgene expression is modulated relative to an AAV vector comprising an AAV genome with a wild-type MAAP encoding sequence.

5. The method of any one of claims 1-4, wherein the mutated MAAP encoding sequence comprises an insertion encoding one or more amino acids.Attorney Docket No.5470.975.WO 6. The method of claim 5, wherein the mutated MAAP encoding sequence comprises an insertion in the AAV genome encoding 6 glutamates or 6 alanines.

7. The method of any one of claims 1-4, wherein the mutated MAAP encoding sequence comprises a mutation in the start codon of MAAP.

8. The method of any one of claims 1-4, wherein the mutated MAAP encoding sequence comprises a mutation of one or more codons, optionally the start codon.

9. The method of any one of the previous claims, wherein the mutated MAAP encoding sequence comprises a deletion of one or more codons.

10. The method of any one of the previous claims, wherein the MAAP encoding sequence is partially or completely deleted.

11. The method of any one of the previous claims, wherein the AAV genome further comprises a promoter operably linked to the MAAP encoding sequence.

12. The method of claim 11, wherein the promoter is a cell specific promoter.

13. The method of any one of the previous claims, wherein the AAV vector comprises a capsid protein having a tissue-specific tropism.

14. The method of any one of the previous claims, wherein the AAV vector is an AAV1, AAV2, AAV5 or AAV9 serotype.

15. The method of any one of claims 3-14, further comprising contacting the cell with a second AAV vector that does not comprise a mutated MAAP encoding sequence, wherein transgene expression from the second AAV vector is enhanced or modulated relative to the second AAV vector in the absence of the AAV vector comprising a mutated MAAP encoding sequence.Attorney Docket No.5470.975.WO 16. The method of claim 15, wherein the ratio of the second AAV vector to the AAV comprising a mutated MAAP sequence is between 1:10 to 10:

1.

17. The method of any one of the previous claims, wherein the transgene encodes a polypeptide.

18. The method of claim 17, wherein the polypeptide is a therapeutic polypeptide.

19. The method of any one of claims 1-16, wherein the transgene encodes a functional nucleic acid.

20. The method of any one of the previous claims, wherein modulating expression comprises increasing expression of the transgene.

21. The method of any one of the previous claims, wherein modulating expression comprises decreasing expression of the transgene.

22. A nucleic acid encoding an AAV genome comprising a mutated MAAP encoding sequence.

23. The nucleic acid of claim 22, wherein the mutated MAAP encoding sequence comprises a deletion of one or more codons.

24. The nucleic acid of claim 22 or 23, wherein the MAAP encoding sequence is partially or completely deleted.

25. The nucleic acid of claim 22, wherein the mutated MAAP encoding sequence comprises an insertion encoding one or more amino acids.

26. The nucleic acid of claim 25, wherein the mutated MAAP encoding sequence comprises an insertion in the AAV genome encoding 6 glutamates or 6 alanines.Attorney Docket No.5470.975.WO 27. The nucleic acid of claim 22, wherein the mutated MAAP encoding sequence comprises a mutation in a start codon of MAAP.

28. The nucleic acid of claim 27, wherein the start codon of MAAP is mutated from CTG to CCG.

29. The nucleic acid of any one of claims 22-28, wherein the nucleic acid further encodes a promoter operably linked to the MAAP encoding sequence.

30. The nucleic acid of claim 25, wherein the promoter is a cell specific promoter.

31. The nucleic acid of any one of claims 22-30, further comprising a transgene sequence.

32. The nucleic acid of claim 31, wherein the transgene encodes a functional nucleic acid.

33. The nucleic acid of claim 31, wherein the transgene encodes a polypeptide.

34. The nucleic acid of claim 33, wherein the polypeptide is a therapeutic polypeptide.

35. The nucleic acid of any one of claims 22-34, wherein the nucleic acid is a plasmid, phage, viral vector, bacterial artificial chromosome, or yeast artificial chromosome.

36. An AAV particle comprising the nucleic acid of any one of claims 22-35.

37. An AAV vector produced by the method of any one of claims 1-2, 7-14, or 17-19.

38. An AAV genome comprising a mutated MAAP encoding sequence encoded by the nucleic acid of any one of claims 22-35.Attorney Docket No.5470.975.WO 39. A pharmaceutical formulation comprising the nucleic acid of any one of claims 25-36, the AAV particle of claim 36, the AAV vector of claim 37, or the AAV genome of claim 38, in a pharmaceutically acceptable carrier.

Images