Non-neutralizing AAV constructs and methods of generating and using same

Abstract

Provided are engineered AAV vector designs that reduce immunoreactivity and allow for repeated AAV vector injections, and uses thereof alone or in combination with immunosuppressive agents or B cell inhibitors to prevent or evade neutralizing antibodies (NAs).

Description

Attorney Docket No.206193-0124-00WO Non-Neutralizing AAV Constructs and Methods of Generating and Using Same CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 510,917, filed June 29, 2023, which is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION

[0002] Adeno-associated viruses (AAVs) are apathogenic dependoviruses that in humans, upon infection with a helper virus, persist for the lifetime of the individual. (Berns KI, et al., 2017, Hum Gene Ther, 28:308-313; Hüser D, et al., 2017, J Virol, 9:e02137-16). It was initially assumed that gene transfer with AAV vectors would be tolerated by humans without eliciting destructive immune responses or adverse events (AE). This assumption was invalidated in early clinical trials for hemophilia B where AAV vectors were shown to stimulate AAV capsid-specific T cells resulting in a mild transaminitis and loss of transgene product expression. (Friedman-Einat M, et al., 1997, J Clin Microbiol, 35:71-8. Manno CS, et al., 2006, Nat Med, 12:342-7). The initial vector dose of 2 x 1012 viral genomes (vg) / kg used for correction of hemophilia B was relatively modest and AAV-specific T cell responses and the associated subclinical liver damage could be controlled with transient immunosuppression with steroids. (Mingozzi F, et al., Nat Med, 2007;13:419-22). Switching to AAV vectors that express the gain-of- function coagulation factor (F)IX Padua mutant allowed for a 4-fold reduction of the therapeutic AAV vector dose, which in most patients ameliorated the need for immunosuppression. (Nathwani AC, et al., 2011, N Engl J Med, 365:2357-65). These early results highlight the strong relationship between vector dose and immunotoxicity.

[0003] Subsequent trials for correction of CNS or neuromuscular disorders escalated the dose of AAV vectors to >1014 vg / kg to achieve therapeutic benefits. (George LA, et al., 2017, N Engl J Med, 377:2215-2227; Asher DR, et al., 2020, Expert Opin Biol Ther, 20:263-274). These large doses in turn triggered in some patients even if they were concomitantly treated with immunosuppressants serious and occasionally even fatal AEs. (Jablonka S, et al., 2022, Neurol Res Pract, 4:2). Serious adverse events(SAEs) included liver damage in patients who received 1.1 x 1014 vg / kg of onasemnogene, an FDA-approved AAV9 vector for treatment of spinal muscular atrophy (SMA). (High-dose AAV gene therapy deaths, 2020, Nat Biotechnol, 38:910) Similarly, in a trial for treatment of X-linked myotubular myopathy (XLMTM) 3 out of 17 patients in the 3.5 x 1014 vg / kg AAV vector dose group developed fulminant hepatitis and died despite immunosuppression. (Jablonka S, et al., 2022, Neurol Res Pract, 4:2; Chand D, et al., 2021, J Hepatol, 74:560-566). After the initial FDA hold was lifted the next patient, who received a reduced dose of 1.3 x 1014 vg / kg, developed signs of hepatic disease, and died. (Mullard A.2021, Nat Rev Drug Discov, 20:804-805). Clinical trials for Duchenne muscular dystrophy (DMD) observed myocarditis after transfer of AAV vectors at doses of 2 x 1014 vg / kg. (Mavrogeni S, et al., 2010, BMC Neurol, 10:33). AAV vector doses equal or exceeding 5 x 1013 vg / kg resulted in 9 out of 1400 SMA patients in thrombotic microangiopathy (TMA), which due to clot formation within capillaries led to kidney damage. Similarly, TMA was observed in 4 out of 15 boys with DMD who received AAV vectors. (Guillou J, et al., 2022, Blood Adv, 6:4266–4270). TMA was not only observed in children but also in adults treated for Fabry disease, Danon disease or Methylmalonic Acidemia. One subject who received an intrathecal injection of 3.5 x 1013 vg of an AAV9 vector for treatment of giant axonal neuropathy and one patient who received by the same route 4.2 x 1014 vg of an AAVrh10 vector for correction of amyotrophic lateral sclerosis developed symptoms indicative for DRG toxicity and both were found to have suffered neuronal loss at autopsy. (Jablonka S, et al., 2022, Neurol Res Pract, 4:2; Mavrogeni S, et al., 2010, BMC Neurol, 10:33; Buss N, et al., 2022, Mol Ther Methods Clin Dev, 24:342-354).

[0004] As such, there is a need in the art for compositions and methods that allow for a shift from toxic single-dose injections of AAV vectors. The present invention satisfies this unmet need. SUMMARY OF THE INVENTION

[0005] The present invention is partly based on the development of engineered AAV constructs with reduced immunogenicity. In some embodiments, the engineeredAAV is administered at low doses. In some embodiments, the engineered AAV is administered in a single dose or multiple doses.

[0006] The present invention is also partly based on the development of drug combinations that reduce B-cell response to AAV administration. In some embodiments, the drug combination allows for single-dose or repeated-dose injections of AAV vectors.

[0007] In one embodiment, the invention relates to a composition comprising an engineered adeno-associated virus (AAV) virion, wherein the AAV capsid comprises one or more amino acid mutations that inhibits its binding to C3b, or a fragment thereof.

[0008] In one embodiment, the AAV is AAV3.

[0009] In one embodiment, the AAV virion comprises a nucleic acid molecule comprising a heterologous nucleotide sequence encoding a therapeutic agent flanked by AAV ITRs. In one embodiment, the heterologous nucleotide sequence comprises a sequence for insertion into the genome of a target cell.

[0010] In one embodiment, the invention relates to a composition comprising a nucleic acid molecule, or combination of nucleic acid molecules, comprising the set of genes for generation of an engineered adeno-associated virus (AAV) virion, wherein the AAV capsid comprises one or more amino acid mutations that inhibits its binding to C3b, or a fragment thereof.

[0011] In one embodiment, the nucleic acid molecule, or combination of nucleic acid molecules, comprises at least one nucleotide sequence encoding an engineered AAV capsid comprising one or more amino acid mutations that inhibits its binding to C3b, or a fragment thereof, and further comprises at least one transgene nucleotide sequence flanked by AAV ITRs.

[0012] In one embodiment, the invention relates to a method of gene therapy comprising administering a composition comprising an engineered adeno-associated virus (AAV) virion, wherein the AAV capsid comprises one or more amino acid mutations that inhibits its binding to C3b, or a fragment thereof, to a subject in need thereof. In one embodiment, the AAV is AAV3.

[0013] In one embodiment, the AAV virion comprises a nucleic acid molecule comprising a heterologous transgene nucleotide sequence flanked by AAV ITRs. In oneembodiment, the heterologous transgene nucleotide sequence is inserted into the genome of a target cell following administration of the composition.

[0014] In one embodiment, the composition is administered more than once.

[0015] In one embodiment, the composition is administered in combination with at least one additional agent.

[0016] In one embodiment, the composition is administered in combination with a helper virus or helper vector.

[0017] In one embodiment, the composition is administered in combination with an immunosuppressive agent.

[0018] In one embodiment, the composition is administered in combination with a B cell inhibitor.

[0019] In one embodiment, the composition is administered in combination with a C3 complement fragment, Fostamatinib, Ibrutinib, Idelalisib or Bortezomib.

[0020] In one embodiment, the gene therapy is administered for the treatment of Duchenne muscular dystrophy (DMD), thalassemia, sickle cell disease, cystic fibrosis, polycystic kidney disease, or Tay–Sachs disease.

[0021] In one embodiment, the invention relates to a method of gene therapy comprising administering a composition comprising a nucleic acid molecule, or combination of nucleic acid molecules, comprising at least one nucleotide sequence encoding an engineered AAV capsid comprising one or more amino acid mutations that inhibits its binding to C3b, or a fragment thereof, and further comprising at least one transgene nucleotide sequence flanked by AAV ITRs, to a subject in need thereof. In one embodiment, the AAV is AAV3. In one embodiment, the heterologous transgene nucleotide sequence is inserted into the genome of a target cell following administration of the composition.

[0022] In one embodiment, the composition is administered more than once.

[0023] In one embodiment, the composition is administered in combination with at least one additional agent.

[0024] In one embodiment, the composition is administered in combination with a helper virus or helper vector.

[0025] In one embodiment, the composition is administered in combination with an immunosuppressive agent.

[0026] In one embodiment, the composition is administered in combination with a B cell inhibitor.

[0027] In one embodiment, the composition is administered in combination with a C3 complement fragment, Fostamatinib, Ibrutinib, Idelalisib or Bortezomib.

[0028] In one embodiment, the gene therapy is administered for the treatment of Duchenne muscular dystrophy (DMD), thalassemia, sickle cell disease, cystic fibrosis, polycystic kidney disease, or Tay–Sachs disease.

[0029] In one embodiment, the invention relates to a method of reducing or preventing the generation of neutralizing antibodies (NAs) in a subject, the method comprising administering at least one B-cell inhibitor.

[0030] In one embodiment, at least one B-cell inhibitor is administered in combination with at least one AAV virion. In one embodiment, at least one B-cell inhibitor is administered in combination with at least one nucleic acid molecule comprising the set of genes for the generation of at least one AAV virion.

[0031] In one embodiment, the B-cell inhibitor is a C3 complement fragment, Fostamatinib, Ibrutinib, Idelalisib or Bortezomib. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following detailed description of embodiments of the invention can be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0033] Figure 1 illustrates fragments of C3 and their location (numbers) within the protein. The thioester within C3d is highlighted. ….S-S bonds.

[0034] Figure 2 illustrates simplified signaling pathways that trigger B cell activation. Lead drugs for testing are highlighted in red but for APL-9 which does not inhibit C3 activation in mice.

[0035] Figure 3, comprising Figure 3A to Figure 3C, depicts results demonstrating the effect of rapamycin and ibrutinib on B cell responses and Abproduction. (Figure 3A) Mice were treated for 21 days with 1.5 mg / kg of rapamycin (R) i.p., given daily alone or with Ibrutibib (I) at 25 mg / kg per oz. Mice were injected with 5 x 1010vg of a CpG reduced AAV8-LacZ vector 7 days after onset of drug treatment. They were injected with an CpG unmodified AAV8-LacZ vector 8 weeks later. Mice were bled periodically and AAV Ab titers were analyzed by an ELISA using individual sera diluted to 1:100. (Figure 3B) Mice were treated with drugs as in (Figure 3A) and injected with 5 x 1010vg of an AAVrh32 / 33-LacZ vector. Mice were bled and analyzed as in (Figure 3A). (Figure 3C) Mice were injected with the equivalent of 109vg of an empty AAV3 vector. Drug treatment was initiated 3 weeks later. Mice were immunized one week later, and their sera were analyzed as in (Figure 3A) using individual sera at a 1:400 dilution. Data show results of individual sera, bars show medians, stars above indicate significant differences by 2-way Anova. (*) – p value between 0.01-0.05, (**) p value between 0.001 to 0.01, (***) p value between 0.0001 and 0.001, (****) p value <0.0001.

[0036] Figure 4 is a table of drug regimens that block induction of AAV NAs to allow for repeated dosing of an AAV3-hF.IX vector.

[0037] Figure 5, comprising Figure 5A and Figure 5B, illustrates a (Figure 5A) model of iC3b bound to AAV2 caspid (Figure 5A), including a zoom into the binding site (Figure 5B).

[0038] Figure 6 shows to the right the iC3b alpha chain in grey; the domain that is expected to bind to AAV capsid and that will be deleted to confirm the model shown in Figure 5 is shown in black. The sequence of the C3b alpha chain is provided in SEQ ID NO:1 highlighting the sequence that will be deleted (SEQ ID NO:2). SEQ ID NO:3 (underlined) reflects a fragment C3f (Figure 1) that has been deleted from C3b to form iC3b. DETAILED DESCRIPTION

[0039] The invention partly relates to the development of regimens that block B cell responses to adeno-associated viral (AAV) vectors to allow for a shift from toxic single-dose injections to repeated treatments with more moderate and safe doses. In one embodiment, the invention includes AAV vectors with capsid modifications within thebinding site for the complement component iC3b, which prevents binding of the AAV capsid to complement receptors on follicular dendritic cells and decreases B cell responses.

[0040] The invention partly relates to combinations of adeno-associated viruses (AAVs) and therapeutic agents that reduce the production of neutralizing antibodies (NAs) when administered. The invention further relates to AAV modifications that allow for single-dose or multiple-dose AAV injections. The invention further relates to drug combinations that inhibit primary and recall B cell responses. Definitions

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary 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.

[0042] As used herein, each of the following terms has the meaning associated with it in this section.

[0043] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0044] AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.

[0045] “AAV helper functions” refer to AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. Thus, AAV helper functions include both of the major AAVopen reading frames (ORFs), rep and cap. The Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking ofthe AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. The Cap expression products supply necessary packaging functions. AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vectors.

[0046] The term “AAV helper construct” refers generally to a nucleic acid molecule that includes nucleotide sequences providing AAV functions deleted from an AAV vector which is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest. AAV helper constructs are commonly used to provide transient expression of AAV rep and / or cap genes to complement missing AAV functions that are necessary for lytic AAV replication; however, helper constructs lack AAV ITRs and can neither replicate nor package themselves. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs and vectors that encode Rep and / or Cap expression products have been described. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol.65:2936-2945.

[0047] The term “accessory functions” refers to non-AAV derived viral and / or cellular functions upon which AAV is dependent for its replication. Thus, the term captures proteins and RNAs that are required in AAV replication, including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.

[0048] The term “accessory function vector” refers generally to a nucleic acid molecule that includes nucleotide sequences providing accessory functions. An accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virion production in the host cell.

[0049] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0050] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0051] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0052] The term “engineered AAV vector” means a vector derived from an adeno-associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, and comprising one or more insertion, deletion, or substitution in one or more structural protein.

[0053] By “recombinant virus” is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.

[0054] By “AAV virion” is meant a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat). In this regard, single-stranded AAV nucleic acid molecules of either complementary sense, e.g., “sense” or “antisense” strands, can be packaged into any one AAV virion and both strands are equally infectious.

[0055] A “recombinant AAV virion,” or “rAAV virion” is defined herein as an infectious, replication-defective virus including an AAV protein shell, encapsidating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs. A rAAV virion is produced in a suitable host cell which has had an AAV vector, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0056] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a DNA, or an RNA, to serve as templatesfor synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0057] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0058] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0059] The term “host cell” denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an AAV helper construct, an AAV vector plasmid, an accessory function vector, or other transfer DNA. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

[0060] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal or cell whether in vitro or in vivo, amenable to the methods described herein. In one embodiment, the subjects includevertebrates and invertebrates. Invertebrates include, but are not limited to, Drosophila melanogaster and Caenorhabditis elegans. Vertebrates include, but are not limited to, primates, rodents, domestic animals or game animals. Primates include, but are not limited to, chimpanzees, cynomologous monkeys, spider monkeys, and macaques (e.g., Rhesus). Rodents include, but are not limited to, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), and fish (e.g., zebrafish, trout, catfish and salmon). In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0061] By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil). The term “nucleic acid” typically refers to large polynucleotides.

[0062] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0063] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0064] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0065] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced substantially only when an inducer which corresponds to the promoter is present.

[0066] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0067] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a conditional manner.

[0068] As used herein, “operably linked” sequences include both expression control sequences that are contiguous with a sequence encoding a molecule of interest and expression control sequences that act in trans or at a distance to control the sequence encoding a molecule of interest.

[0069] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0070] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0071] The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. Such techniques can be used to introduce one or more exogenous DNA moieties, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

[0072] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. Description

[0073] The present invention generally relates to compositions and methods to deliver AAVs while reducing SAEs. In one embodiment, the invention relates to an engineered AAV modified to not bind to or otherwise have a reduced affinity to bind to the complement C3. In one embodiment, the invention relates to a combination of drugs that inhibit primary and recall B cell responses. Engineered AAV Vectors

[0074] Central to the present invention is the discovery that the AAV capsid interaction with to complement factor C3 products, such as iC3b, allows for deposition of AAV particles onto complement receptors on follicular dendritic cells (FDCs), which in turn drives activation of germinal center (GC) B cells and further binding of AAVparticles to CD21 which amplifies B cell receptor signaling. Therefore, in part, the invention relates to compositions and methods that reduce or inhibit binding of the AAV capsid to complement factor C3 products, including iC3b. In some embodiments, provided are engineered AAV vectors encoding a variant AAV virion comprising at least one mutation in the AAV capsid which prevents binding of the AAV capsid to complement factor C3 products, including iC3b.

[0075] In one embodiment, the present invention relates to a composition comprising an engineered variant of an adeno-associated virus (AAV) vector (i.e. an engineered AAV vector). AAV vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for delivery and expression of DNA encoding therapeutic molecules, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a DNA sequence encoding a therapeutic molecule contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, delivery method.

[0076] In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). The term "AAV vector" means a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, and AAV-9. AAV vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

[0077] AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Despite the high degree of homology, the different serotypes have tropisms for different tissues. The receptor for AAV1 is unknown; however, AAV1 is known to transduce skeletal and cardiac muscle more efficiently than AAV2. Since most of thestudies have been done with pseudotyped vectors in which the vector DNA flanked with AAV2 ITR is packaged into capsids of alternate serotypes, it is clear that the biological differences are related to the capsid rather than to the genomes. Recent evidence indicates that DNA expression cassettes packaged in AAV 1 capsids are at least 1 log 10 more efficient at transducing cardiomyocytes than those packaged in AAV2 capsids.

[0078] In one embodiment, provided is an adeno-associated viral delivery system. The adeno-associated virus can be of serotype 1 (AAV 1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9).

[0079] Desirable AAV fragments for assembly into vectors include the cap proteins, including the vp1, vp2, vp3 and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells. Such fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, without limitation, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. An artificial AAV serotype may be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. In some embodiments, the engineered AAV vector encodes an AAV virion that lacks the ability to bind to complement C3 or the complement C3 cleavage product iC3b.

[0080] In one embodiment, the engineered AAV vector comprises an engineered AAV3 vector. In some embodiments, the engineered AAV3 vector encodes a virion comprising one or more amino acid substitution, deletion or insertion in the natural (i.e. wild-type) AAV3 viral capsid. In some embodiments, the engineered AAV3 virion lacks the ability to bind to complement C3 or the complement C3 cleavage product iC3b.

[0081] The AAV mutants of the subject invention preferably display decreased immunoreactivity relative to neutralizing antibodies that may be present in the host to which the mutant virions are administered. In this way, cells and tissues of subjects that have either been naturally infected with AAV (i.e., due to previous natural infection) or artificially infected with AAV (i.e., due to previous gene therapy or nucleic acid immunization) may be more efficiently transfected with recombinant AAV virions in order to treat or prevent new or on-going disease.

[0082] The modified AAV vectors of the present invention may be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos.5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therapy vector.

[0083] Alternatively, AAVs may be mutated such that neutralizing antibodies are unable to bind to the AAV virion. A well-studied mechanism for neutralization is that a neutralizing antibody physically blocks a region on the virus required to bind to receptors that are required for infection. Previous studies with other viruses have shown that the receptors and neutralizing antibodies bind to a distinct set of amino acids and that it is possible to identify mutants at particular positions on viral capsids that affect the binding of neutralizing antibodies, but not receptors or other functions needed for viral infection. Experiments in which wild-type replicating viruses are selected to be resistant to neutralizing antibodies have shown that mutations, even in single, double, triple, quadruple or more amino acids, such as those described here, can result in significant increases in resistance to antibody neutralization.

[0084] The ability or inability of an AAV mutant virion to bind AAV antisera is partially a function of the sequence of the capsid proteins (encoded by AAV cap gene). Thus, the invention contemplates single, double, triple, quadruple and more amino acid changes made on the surface of the AAV virion, as well as deletions and / or insertions, in order to decrease immunoreactivity, e.g., to alter the ability of the AAV virion to bind AAV antisera. Such mutants may be assessed for resistance to neutralization and, if necessary, more drastic or multiple changes can be made.

[0085] Methods of identifying portions of the AAV virion amenable to mutation with a resulting functional AAV virion are described in the examples below. Generally, the naturally occurring amino acid is substituted with an amino acid that has a small side- chain and / or is uncharged and is therefore less immunogenic. Such amino acids include, without limitation, alanine, valine, glycine, serine, cysteine, proline, as well as analogs thereof, with alanine preferred. Moreover, additional mutations can be present.

[0086] The various amino acid positions occurring in the capsid protein are numbered herein with reference to the AAV3 sequence. However, it is to be understood that mutations of amino acids occurring at corresponding positions in any of the AAV serotypes are encompassed by the present invention.

[0087] Based on the above discussion, and the examples provided herein, one of skill in the art can reasonably predict mutations that can be made to sequences of wild- type AAV and currently used AAVs for gene therapy in order to generate AAV virions with decreased immunoreactivity. Based on the knowledge provided by the cryo-EM of AAV3-iC3b complexes, other AAV serotypes can be mutated and used with reduced immunoreactivity as defined above.

[0088] In one embodiment, the engineered AAV vector of the present invention comprises a modified viral genome. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part (e.g., the rep and / or cap genes), but retain functional flanking inverted terminal repeats (ITRs) that fold into characteristic T-shaped hairpins, which are the only sequences required in cis for the replication and packaging of the AAV genome. In some embodiments, the deleted wild-type genes can be replaced by a sequence that enables expression of one or more therapeutic molecule. In one embodiment, said sequence comprises conventional control elements which are operably linked to a sequence encoding a therapeutic molecule in a manner which permits its transcription, translation and / or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; andwhen desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0089] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function either cooperatively or independently to activate transcription.

[0090] One example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor -1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. It should be understood that the examples below demonstrate the use of a hepatocyte-specific promoter, but this invention is not limited to such promoter.

[0091] Promoters can also be used to restrict expression of a therapeutic molecule of interest to a particular cell-type, and thus reduce or prevent off-target effects of expression in undesired cell-types. In one embodiment of the present invention, the modified viral genome comprises a neural cell-specific promoter. Neural cell-specific promoters include, but are not limited to, synapsin I (Syn) specific for neurons, glial fibrillary acidic protein (GFAP) specific for astrocytes, myelin basic protein (MBP)specific for oligodendrocytes, and CD68 specific for microglia. In one embodiment, the neural cell-specific promoter limits expression to one or more cell selected from the group consisting of, but not limited to, neurons, astrocytes, oligodendrocytes, and microglia. In one embodiment, the neural cell-specific promoter is one or more selected from the group consisting of: Syn, GFAP, MBP, and CD68.

[0092] Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0093] Enhancer sequences found on a vector also regulate expression of the sequence of interest contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector. Methods of Production

[0094] In one embodiment, the present invention comprises at least one engineered AAV vector that results in generation of modified AAV virions which evade the immune systems, and therefore elicit low or no production of neutralizing antibodies against the AAV virion. In one embodiment, method comprises: 1) transfecting a suitable host cell with at least one nucleic acid molecule comprising a modified AAV viral genome wherein at least one viral gene is replaced by at least one nucleic acid molecule for delivery to a target cell and a nucleotide sequence encoding the engineered AAV vector viral capsid, and 2) isolating the packaged engineered AAV viral vector from the suitable host cell. Exemplary host cells include, but are not limited to, mammalian cells and insect cells. In some embodiments, the method further comprises transfecting saidsuitable host cell one with or more helper plasmids encoding at least one protein required for AAV replication, at least one adenovirus helper gene, or a combination thereof. In some embodiments, the method further comprises transfecting said suitable host cell with one or more nucleic acid encoding at least one therapeutic agent. In some embodiments, the modified AAV viral genome has been modified to replace at least one viral gene with a nucleotide sequence encoding a therapeutic agent for delivery by the modified AAV virion to a target cell of interest. Exemplary therapeutic nucleic acid molecules that can be delivered using the modified AAV virions include, but are not limited to a gene therapy agent, gene editing agent (e.g., CRISPR), an antisense nucleic acid molecule (e.g., sgRNA, siRNA, miRNA, etc.), or any combination thereof. In some embodiments, the nucleotide sequence encoding a therapeutic agent for delivery by the modified AAV virion is flanked by AAV ITRs to promote packaging of the nucleotide sequence into AAV particles.

[0095] In some embodiments, nucleotide sequences encoding a) the modified AAV viral genome comprising a replacement of at least one viral gene with a nucleic acid molecule for delivery by the modified AAV virion to a target cell of interest (e.g., gene therapy agent) flanked by AAV ITRs, b) the engineered AAV vector viral capsid, c) at least one protein required for AAV replication, and d) at least one nucleic acid encoding one or more adenovirus helper gene are contained within the same vector. In some embodiments, the nucleic acid comprising the modified AAV viral genome is contained within a first vector, the nucleic acid encoding the engineered AAV vector viral capsid and the one or more nucleic acid encoding proteins required for AAV replication are contained within a second vector, and the one or more nucleic acid encoding one or more adenovirus helper gene are contained within a third vector.

[0096] Methods of preparing engineered AAV-based vectors and other AAV vectors as are described herein are known. The sequences of any of the AAV capsids provided herein can be readily generated using a variety of techniques. Suitable production techniques are well known to those of skill in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively, peptides can also be synthesized by the well-known solid phase peptide synthesis methods (Merrifield, (1962) J. Am. Chem. Soc., 85:2149;Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62). These and other suitable production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.

[0097] The components required to be cultured in the host cell to package a modified viral genome in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., modified viral genome, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the modified viral genome. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contains the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0098] The modified AAV viral genome, rep sequences, cap sequences, and helper functions required for producing the engineered AAV vector of the invention may be delivered to the packaging host cell in the form of any genetic element which transfer the sequences carried thereon. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating AAV virions are well known and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al, (1993) J. Virol., 70:520-532 and U.S. Pat. No.5,478,745.

[0099] Unless otherwise specified, the AAV ITRs, and other selected AAV components described herein, may be readily selected from among any AAV. These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from an AAV sequence. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank®, PubMed®, or the like.

[0100] The modified AAV viral genome can be carried on any suitable vector, e.g., a plasmid, which is delivered to a host cell. The plasmids useful in this invention may be engineered such that they are suitable for replication and, optionally, integration in prokaryotic cells, mammalian cells, or both. These plasmids (or other vectors carrying the 5′ AAV ITR-heterologous molecule-3′ AAV ITR) contain sequences permitting replication of the modified AAV viral genome in eukaryotes and / or prokaryotes and selection markers for these systems. Selectable markers or reporter genes may include sequences encoding geneticin, hygromicin or purimycin resistance, among others. The plasmids may also contain certain selectable reporters or marker genes that can be used to signal the presence of the vector in bacterial cells, such as ampicillin resistance. Other components of the plasmid may include an origin of replication and an amplicon, such as the amplicon system employing the Epstein Barr virus nuclear antigen. This amplicon system, or other similar amplicon components permit high copy episomal replication in the cells. Preferably, the molecule carrying the modified AAV viral genome is transfected into the cell, where it may exist transiently. Alternatively, the modified AAV viral genome (carrying the 5′ AAV ITR-heterologous molecule-3′ ITR) may be stably integrated into the genome of the host cell, either chromosomally or as an episome. In certain embodiments, the modified AAV viral genome may be present in multiple copies, optionally in head-to-head, head-to-tail, or tail-to-tail concatamers. Suitable transfection techniques are known and may readily be utilized to deliver the minigene to the host cell. Methods of Administration

[0101] The above-described engineered AAV virion may be delivered to host cells according to published methods. The engineered AAV virion, preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

[0102] Optionally, the compositions of the invention may contain, in addition to the engineered AAV virion and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0103] The engineered AAV virion is administered in sufficient amounts to transfect the cells and to provide sufficient levels of therapeutic molecule expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., the lung, heart, or brain), oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired.

[0104] Delivery of the AAV virions to a mammalian subject may be by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit the mutant virions into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the AAV virions. Moreover, for certain conditions, it may be desirable to deliver themutant virions to the CNS of a subject. By “CNS” is meant all cells and tissue of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like. AAV virions or cells transduced in vitro may be delivered directly to the CNS or brain by injection into, e.g., the ventricular region, as well as to the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobule, with a needle, catheter or related device, using neurosurgical techniques known in the art, such as by stereotactic injection. Methods of Treatment

[0105] In some embodiments, the compositions of the inventions are used for gene therapy. In some embodiments, gene therapy is beneficial for the treatment of a monogenic disease or disorder, wherein the AAV viral genome is replaced with a heterologous nucleotide sequence encoding a gene that is missing or deficient in a subject. Examples of monogenic diseases include, but are not limited to, Duchenne muscular dystrophy (DMD), thalassemia, sickle cell disease, cystic fibrosis, polycystic kidney disease, and Tay–Sachs disease.

[0106] The dose of AAV virion required to achieve a particular “therapeutic effect,” e.g., the units of dose in vector genomes / per kilogram of body weight (vg / kg), will vary based on several factors including, but not limited to: the route of AAV virion administration, the level of gene (or anti-sense RNA or ribozyme) expression required to achieve a therapeutic effect, the specific disease or disorder being treated, a host immune response to the AAV virion, a host immune response to the gene (or anti-sense RNA or ribozyme) expression product, and the stability of the gene (or anti-sense RNA or ribozyme) product. One of skill in the art can readily determine a AAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.

[0107] Generally speaking, by “therapeutic effect” is meant a level of expression of one or more heterologous nucleic acids containing a therapeutic protein sufficient to alter a component of a disease (or disorder) toward a desired outcome or clinical endpoint, such that a patient's disease or disorder shows clinical improvement, oftenreflected by the amelioration of a clinical sign or symptom relating to the disease or disorder.

[0108] The constructs of the present invention may alternatively be used to deliver heterologous nucleic acids encoding a protein of interest to a host cell in order to elucidate its physiological or biochemical function(s). The heterologous nucleic acid can be either an endogenous gene or heterologous. Using either an ex vivo or in vivo approach, the skilled artisan can administer the mutant virions containing one or more heterologous nucleic acid of unknown function to an experimental animal, express the heterologous nucleic acid(s), and observe any subsequent functional changes. Such changes can include: protein-protein interactions, alterations in biochemical pathways, alterations in the physiological functioning of cells, tissues, organs, or organ systems, and / or the stimulation or silencing of gene expression.

[0109] Alternatively, the skilled artisan can of over-express a gene of known or unknown function and examine its effects in vivo. Such genes can be either endogenous to the experimental animal or heterologous in nature (i.e., a transgene).

[0110] By using the methods of the present invention, the skilled artisan can also abolish or significantly reduce gene expression, thereby employing another means of determining gene function.

[0111] The methods of the present invention are compatible with other well- known technologies such as transgenic mice and knockout mice and can be used to complement these technologies. One skilled in the art can readily determine combinations of known technologies with the methods of the present invention to obtain useful information on gene function

[0112] In some embodiments, the present invention comprises methods of promoting regeneration of damaged or degenerated neural tissue in a subject in need thereof. In some embodiments, the method comprises administering to a subject having damaged or degenerated tissue a composition comprising the engineered AAV vector of the present invention.

[0113] In some embodiments, the composition is administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

[0114] In some embodiments, the viral genome (vg) concentration of the composition that is administered is between 1.0×1011vg per kilogram (kg) and 1.0×1016vg / kg. In some cases, the concentration of infectious particles of at least or about 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017. In some cases the concentration of infectious particles is 2×107, 2×108, 2×109, 2×1010, 2×1011, 2×1012, 2×1013, 2×1014, 2×1015, 2×1016, or 2×1017. In some cases the concentration of the infectious particles 3×107, 3×108, 3×109, 3×1010, 3×1011, 3×1012, 3×1013, 3×1014, 3×1015, 3×1016, or 3×1017. In some cases the concentration of the infectious particles 4×107, 4×108, 4×109, 4×1010, 4×1011, 4×1012, 4×1013, 4×1014, 4×1015, 4×1016, or 4×1017. In some cases the concentration of the infectious particles 5×107, 5×108, 5×109, 5×1010, 5×1011, 5×1012, 5×1013, 5×1014, 5×1015, 5×1016, or 5×1017. In some cases the concentration of the infectious particles 6×107, 6×108, 6×109, 6×1010, 6×1011, 6×1012, 6×1013, 6×1014, 6×1015, 6×1016, or 6×1017. In some cases the concentration of the infectious particles 7×107, 7×108, 7×109, 7×1010, 7×1011, 7×1012, 7×1013, 7×1014, 7×1015, 7×1016, or 7×1017. In some cases the concentration of the infectious particles 8×107, 8×108, 8×109, 8×1010, 8×1011, 8×1012, 8×1013, 8×1014, 8×1015, 8×1016, or 8×1017. In some cases the concentration of the infectious particles 9×107, 9×108, 9×109, 9×1010, 9×1011, 9×1012, 9×1013, 9×1014, 9×1015, 9×1016, or 9×1017.

[0115] In some embodiments, the administering of step is performed once. Alternatively, the administering of step is repeated at least twice. The administering of step may be performed once daily. In some cases, the administering of step comprises intravenous administration. In some cases, the administering comprises pulmonary administration. In some cases, the administering comprises intranasal administration (such as a spray). In some cases, the administering of step comprises injecting the composition into a target in vivo environment. In some cases, the administering of step does not comprise injecting the composition into the target in vivo environment.

[0116] In some embodiments, the subject is a mammal. Non-limiting examples of a mammal include a mouse, rat, guinea pig, rabbit, chimpanzee, or farm animal. In some instances, the mammal is a non-human primate. In some instances, the subject is human. The subject of the present disclosure may not be diagnosed with a disease or condition. Alternatively, the subject may be a patient that is diagnosed with a disease or disorder, or suspected of having the disease or the disorder.

[0117] In some embodiments, the present invention comprises methods of treating or preventing one or more disease or disorder associated with damaged or degenerated neural tissue in a subject. In some embodiments, the method comprises administering to a subject having a disease or disorder associated with damaged or degenerated neural tissue a composition comprising the engineered AAV vector of the present invention. EXPERIMENTAL EXAMPLES

[0118] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0119] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure. Example 1: Non-Neutralizing AAV Constructs and Methods of Generating and Using Thereof

[0120] Regimens that block B cell responses to adeno-associated viral (AAV) vectors were developed to allow for a shift from toxic single-dose injections to repeated treatments with more moderate and safe doses. Challenges imposed by the immunesystem limit the tolerability of AAV gene transfer. The very high doses of AAV vectors of > 1014viral genomes (vg) / kg used for some disorders have resulted in serious adverse events (SAE) or even deaths demonstrating that AAV vector doses that can safely be injected into patients are limited and for some indications below the therapeutic dose. Some of the SAEs, such as myocarditis and thrombotic microangiopathy (TMA) are immune mediated while the etiology of others such as dorsal root ganglion (DRG) toxicity remains unknown. Currently used immunosuppressive drugs have not prevented the SAEs indicating that it may be prudent to treat patients with repeated transfer of moderate doses rather than a single injection of high doses of AAV vectors. The former approach has been avoided as AAV vectors elicit neutralizing antibodies (Abs) (NAs) that prevent successful reapplication of serologically cross-reactive vectors. The use of drugs that target crucial steps of B cell activation signaling cascades was tested.

[0121] Drugs that inhibit Burton Tyrosine Kinase (BTK) and mTOR block primary B cell responses to AAV but fail to inhibit memory B cell development or responses. Additional drugs were explored including those that either target other B cell signaling molecules such as SYK or the p110d chain of PI3K or selectively destroy Ab secreting cells such as brotezimib that by inhibiting the 26S proteasome prevents degradation of misfolded proteins leading to endoplasmic reticulum (ER) stress and plasma cell (PC) death.

[0122] AAV particles induce exceptionally high and sustained Ab responses in part due to the binding of AAV capsid to complement factor C3 products such as iC3b. This allows for deposition of AAV particles onto complement receptors on follicular dendritic cells (FDCs), which in turn are essential to drive activation of germinal center (GC) B cells. It also allows for binding of the iC3b bound AAV particles to CD21 on the surface of B cells, which amplifies B cell receptor signaling. The effect of C3 inhibition on magnitude and longevity AAV-specific B cell responses and their susceptibility to drug treatments was tested. In addition, cryo-electron microscopy (EM) was used to pinpoint the binding sites between iC3b and the AAV capsid. Based on these data AAV vectors with mutations in the iC3b binding site were generated. AAV vectors lacking the iC3b binding was selected and tested to determine changes in immunogenicity. This mutated vector was tested in combination with drugs that inhibit B cell activation. Studieswere conducted in a preclinical mouse model using mainly AAV3 vectors expressing human (h) coagulation factor (F)IX to allow for easy tracking of transduction efficiency.

[0123] The results presented herein identified drug combinations and AAV vectors designs that reduce immunogenicity to AAVs and allow for repeated use of AAV vectors for gene transfer.

[0124] The results of the experiments in this experimental example are now described. Drug combinations on primary and recall B cell responses to an AAV3

[0125] B cells respond to native antigens, which bind to a surface-expressed immunoglobulin (Ig) molecules that serve the B cell receptors (BCR). The BCR on naïve B cells is composed of an IgM molecule while most memory B cells bind antigen through cell surface expressed IgG molecules. BCR signaling is enhanced by antigen bound to CD21 which is expressed on the surface of B cells as a complex with CD19 and CD81. CD21 is a receptor for complement C3 cleavage products (Figure 1) such as the AAV capsid binding iC3b. Antigen bound through C3 components to CD21 can be recognized by the BCR. This results in colocalization of the BCR and the CD19 / CD21-C3- antigen / CD81 complexes into lipid rafts, which in turn delays BCR internalization, causing prolonged and enhanced signaling and thereby higher and broader Ab responses. (Cherukuri A, et al., 2001, Immunity, 14:169-79). The enhancement of Ab responses by C3b fragments had led to the use of, for example, C3d or a peptide that contains the pathogen-binding thioester of C3b (Cerutti A, et al., Nat Rev Immunol.2013;13:118-32. Boothby MR, et al., Genes Dev.2019;33:26-48) as adjuvants for experimental vaccines (Vos Q, et al., 2000, Immunol Rev, 176:154-70; Mesin L, et al., 2016, Immunity, 45(3):471-482; Kranich J, et al., 2016, Front Immunol, 7:225; Mandel TE, et al., 1980, Immunol Rev, 53:29–59).

[0126] Upon encounter with antigen marginal zone (MZ) B cells rapidly start producing IgM Abs. (Xiang Z, et al., 2022, Hum Gene Ther, 33:614-624). This very quick and transient response does not require T cell help. If and to what degree Abs produced by MZ B cells play a role upon AAV gene transfer is unknown. Conventional naïve B cells also called B-2 B cells can either differentiate extrafollicularly into short-lived unswitched or isotype switched plasma cells (PCs) that secrete low affinity IgM or IgG Abs. (Crotty S.2014, Immunity, 41:529-42). Extrafollicular B-2 cell responses to organized highly repetitive forms of antigen such as those present on a viral capsid can be generated without T cell help in presence of innate and BTK signaling (Akkaya M, et al., 2018, Nat Immunol, 19:255-266).

[0127] Alternatively, B cells upon interactions with T helper cells can enter germinal centers (GCs) where they undergo proliferation and affinity maturation within the so-called dark zone (Casola S, et al., 2004, Nat Immunol, 5:317-27). Selection for B cells producing high affinity Abs occurs in the GC’s light zone. There B cells compete for antigen, which they retrieve from follicular dendritic cells (FDCs) (Suan D, et al., 2017, Curr Opin Immunol, 45:97-102). FDCs obtain antigens complexed to Abs or complement factors such as C3 products from marginal zone (MZ) B cells or macrophages through surface expressed Fc or complement receptors. The antigens are then transferred into an intracellular non-degrading compartment and from there periodically cycle back to the cell surface where they become accessible to B cells. Antigen present in or on FDCs can persist for extended periods of time, (Palm AE, et al., 2019, Front Immunol, 10:1787) which could explain rebound Ab responses that were observed in pre-clinical AAV gene transfer trials once treatment with B cell suppressing drugs was stopped (Wen Y, et al., 2019, Blood Sci, :119-129). B cells then process the antigen and present it to TFH cells, (Xu Y, et al., 2014, Cell Res, 24:651-64) an interaction that provides essential B cell survival signals. Processing of antigen by B cells to gain T help is inhibited by TLR9 signaling mediated by CpG motifs (Kehry MR.1996, J Immunol, 156:2345-8) present within the AAV vectors’ genome. B cells with low affinity BCRs are unable to capture antigen for TFH cell interactions and undergo apoptosis. Surviving B cells can exit GCs as memory B cells or Ab secreting PCs, or they can re-enter the dark zones for additional rounds of affinity maturation. These fate decisions are driven by the BCR signaling strength, which regulates different transcription factors (Nelms K, et al., 1999, Annu Rev Immunol, 17:701-38). Memory B cells upon encounter of their cognate antigen can either differentiate directly into terminally differentiated short-lived PCs or their can enter GCs, undergo additional affinity maturation, and develop into long-lived PCs or 2nd generation memory B cells(Konforte D, et al., 2009, J Immunol, 182:1781-7; Xu Y, et al., 2014, Cell Res, 24:651- 64).

[0128] Signaling pathways that regulate B cell differentiation are well known (Figure 2) and offer targets to block Ab responses (Weber MS, et al., 2021, Neurol Neuroimmunol Neuroinflamm, 8:e1067; Sun J, et al., 2017, Hum Gene Ther, 28:654- 666). The IgM BCR expressed by naïve, some memory B and MZ B cells is non- covalently linked to CD79, which is bound to SRC kinases. Upon cross-linking of the BCR changes in the cytoplasmic tail of CD79 lead to activation of spleen tyrosine kinase (SYK). SYK forms a complex with B cell linker protein (BLNK) that activates BTK, and through several additional steps results in activation of p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (Erk)1 / 2 and NF-kB. This is further promoted by signaling through the CD21-CD19-CD81 complex, which lowers the threshold for BCR signaling (Cherukuri A, et al., 2001, Immunity, 14:169-79). CD19 activates PI3Kd, which induces signaling through the mTOR / AKT pathway. Survival signals from TFH cells are provided by CD40-CD40L interactions (Pei X, et al., 2020, Mol Ther Methods Clin Dev, 18:259-268). CD40, through different TRAF adaptor proteins, activates several downstream molecules including PI3K / AKT / PKB, which play a role in cell survival. Cytokines further contribute to B cell activation (El Andari J, et al., 2022, Sci Adv, 8:eabn4704; Asuri P, et al., 2012, Mol Ther, 20:329-38). BCR signaling in class-switched memory B cells that express IgG does not rely on CD79 as the long intracytoplasmic tail of IgG binds directly to SYK (Mingozzi F, et al., 2013, Sci Transl Med, 5:194ra92). BTK is not absolutely required for activation of IgG+ B cells but other yet to be identified factors can compensate (Monteilhet V, et al., 2011, Mol Ther, 19:2084-91).

[0129] It was shown in mice that a combination treatment of rapamycin with ibrutinib, a BTK inhibitor, effectively blocked primary B cell responses (Figure 3A-C), slightly reduced recall responses (Figure 3B), but failed to prevent Ab increases after re- exposure to AAV8 given without drugs (Figure 3A); mice treated only with rapamycin developed Abs after drug-treatment was stopped (rapamycin: Figure 3A wk 6). (Wen Y, et al., 2019, Blood Sci, 1:119-129). Interestingly, inhibition of primary responses by rapamycin and ibrutinib was more complete in mice immunized with highlyimmunogenic AAVrh32 / 33 vectors than with CpG motif reduced AAV8 vectors (Figure 2B).

[0130] Inhibiting B cell responses to AAV vectors to allow for redosing will most likely require a combination of drugs as needs to be explored pre-clinically. Others have succeeded to inhibit primary Ab responses to AAV vectors but once drug treatment was stopped 2-4 weeks after injection of the AAV vector Ab responses rebounded as we also observed in mice treated with rapamycin (Figure 3A). It is possible that this late Ab response is triggered by AAV particles on FDCs and that iC3b play a key role in forming these deposits.

[0131] This study developed drug regimens that blocks induction of AAV NAs to allow for repeated dosing of an AAV3-hF.IX vector. Drugs or combinations of drugs shown in Figure 4 were tested in male mice. Of note female mice show poor transduction with AAV vectors and were therefore not suited for the experiments. Drugs were selected so that in each multiple drug combination, individual drugs target different pathways. Groups of 10 male C57Bl / 6 mice were treated with the various combinations of drugs starting 1 week prior to AAV gene transfer. They then received by i.v. injection 5 x 1010 vg of an AAV3-hF.IX vector, which expresses the transgene under the control of a hepatocyte-specific promoter. Vectors were given again at the same dose 2, 4 and 6 weeks later. Drugs were continued till 2 weeks after the last AAV vector injection. C57Bl / 6 SCID (B6.Cg-Prkdcscid / SzJ) mice (n = 5) which lack functional B and T cells, (Zaiss AK, et al., J Virol.2008;82:2727-40) received the same vector regimen but no drugs. An additional group of C57Bl / 6 mice included in each experiment received the cumulative dose of vector (2 x 1011 vg) once without drugs. Ab responses to AAV was tested 2 weeks after each injection and in addition 6 weeks after the last injection by an ELISA and a virus neutralization assay as described. (Lubinski JM, et al., J Virol.2002 Sep;76(18):9232-41. Kostavasili I, et al., J Immunol.1997 Feb 15;158(4):1763-71) Sera that score positive for Abs were retested to determine the Ab isotypes. hF.IX levels were tested at the same times by an ELISA as described. (Fernández, F.J., et al., Nat Commun 13, 1955 (2022)). Mice were euthanized 8 weeks after the last injection and relative numbers of AAV-specific PCs and memory B cells in spleen and bone marrow were determined by an ELISpot assay. (Alcorlo M, et al., Proc Natl Acad Sci U S A.2011 Aug 9;108(32):13236-40). hF.IX levels in drug-treated mice were compared to those of SCID mice, which are unable to develop Abs, immunocompetent mice, which received a single 2x1011 vg vector dose, and group 1 mice, which received 4 doses of 5 x 1010 vg AAV3 vector without drugs. These comparisons allowed assessment of the effect of spreading the dose of vector over 4 injections (SCID mice receiving 4 doses compared to C57Bl / 5 mice receiving one dose with 4 times the vg content). It further showed if and to what degree inhibition of Abs responses correlates with successful transduction (SCID mice compared to drug treated mice and drug treated mice compared to group 1 mice) and if and to what degree individual drugs or drug combinations affect transduction (hF.IX levels at 2 weeks after the 1st injection in group 1 as compared to drug treated mice tested at the same time). AAV neutralizing Abs were monitored even after completion of gene therapy once a therapeutic dose of transgene product has been achieved to provide further information on immunogenicity of long-term antigen depots. Effects of C3 on B cell responses to an AAV3 vector and the effectiveness of drug treatment.

[0132] The complement factor C3b binds AAV particles where it is then split into iC3b which remains on the particles with high enough avidity to allow for coprecipitation of AAV capsid antigens. (Dunkelberger JR, et al., Cell Res.2010 Jan;20(1):34-50). Ab responses to AAV capsid are reduced (5-10-fold) but not abolished in C3 knockout mice. (Zaiss AK, et al., 2008, J Virol.,82:2727-40) iC3b also binds to the surface of adenovirus and HSV-1. (Smith CJ, et al., 2022, Front Immunol, 13:999021. Majowicz A.2022, AAV Safety Summit). For HSV-1 a domain within the gC protein has been identified to bind to iC3b (Majowicz A.2022, AAV Safety Summit) indicating that the complement factor binds to a specific epitopes rather than through low affinity interactions to multiple sites. The structures of C3, C3b and iC3b have been identified. (Heesters BA, et al., Methods Mol Biol.2017;1623:105-112. Pallesen J, et al., Proc Natl Acad Sci U S A.2017 Aug 29;114(35):E7348-E7357). The 3 proteins have limited flexibility in solution. Cleavage of C3b into iC3b was shown to result in a conformational change which renders iC3b incapable of further activating the complement cascade. (Heesters BA, et al., Methods Mol Biol.2017;1623:105-112). The structure of iC3b bound to its receptors such as CR3has also been resolved. (Pallesen J, et al., Proc Natl Acad Sci U S A.2017 Aug 29;114(35):E7348-E7357).

[0133] Complement has multiple effects on the immune system. (El Andari J, et al., Sci Adv.2022 Sep 23;8(38):eabn4704). It facilitates uptake of pathogens by antigen presenting cells and it triggers pro-inflammatory cytokine responses. (Heesters BA, et al., Methods Mol Biol.2017;1623:105-112). Binding of iC3b-antigen complexes to FDCs is important for induction of GC derived PCs and memory B cells. In the same token iC3b- antigen complexes can bind to CD21 on B cells and from there interact with the BCR and potentiate its signaling up to 100-fold. (Cherukuri A, et al., Immunity.2001;14:169-79). This invention was borne from the hypothesis that C3 inhibition will reduce and shorten Ab responses to AAV by reducing the amount of antigen on FDCs and B cells and that this will enhance the inhibitory effects of drugs that target B cell signaling. Drugs to inhibit human C3 activation are available but they are not effective in mice. Drugs that inhibit C1, which would be expected to reduce C3 activation have limited effects on B cell responses to AAV. Consequently, studies were conducted in C3 knock-out mice and in addition use GFP labeled AAV particles to track their presence over time in lymph nodes.

[0134] In the 1stset of experiments, induction of Abs was tested in groups of 5 male C3 knockout (B6;129S4-C3tm1Crr / J, abbreviated to C3-KO) mice to ensure that C3 plays a role in driving Ab responses to AAV3. C57Bl / 6 mice were used for comparison. Two groups of mice were enrolled. One group of C3-KO mice as well as a group of C57Bl / 6 mice were injected with 2 x 1011vg of an AAV3-hFIX vector. Ab responses and hF.IX levels were tracked in initially in 2 weekly and then in monthly intervals for 4 months. In the 2ndset of experiments, the 2 most effective drug treatments developed in the set of experiments described above were tested in C3-KO mice. The experiment was controlled by C57Bl / 6 mice. Groups of 5 male C3-KO or C57Bl / 6 mice were treated with two combinations of drugs starting 1 week prior to AAV gene transfer. They then received by i.v. injection 5 x 1010vg of an AAV3hF.IX vector, which expresses the transgene under the control of a hepatocyte-specific promoter. Vectors were given again at the same dose 2, 4 and 6 weeks later. Drugs continued till 2 weeks after the last AAV vector injection. Additional groups of C3-KO and C57Bl / 6 mice received the cumulativedose of vector (2 x 1011vg) once or the repeated vector regimen without drugs. Ab and B cell responses and levels of h.FIX expression were monitored as described in the aforementioned experiments till 6 weeks after the last injection of AAV (i.e., 4 weeks after the last drug treatment). Ab responses were expected to be lower and less sustained in C3-KO than in C57Bl / 6 mice resulting in higher efficacy of drug treatments and more importantly in lack of breakthrough Ab response once drug treatment was stopped.

[0135] The next set of experiments tested persistence of AAV particles in normal and C3-KO mice. To this end, an AAV3 vector in which the wild-type VP2 is replaced by a VP2-GFP fusion protein (AAV3-VP2-GFP) was generated using available techniques. To ensure that the VP2 modified AAV still binds iC3b, AAV-VP2-GFP particles were treated for 30 min at room temperature with saline or various concentrations of purified commercially available mouse iC3b. At the same time protein A coated agarose beads were treated with rat IgG1 to mouse C3 / C3b / iC3b (mAb 2 / 11) or an isotype control Ab. Beads were washed and incubated for 1 hour on ice with the serum or C3b pretreated AAV-VP2-GFP particles, washed and analyzed by flow cytometry for presence of GFP+beads. In addition beads treated with AAV3-VP2-GFP or an unmodified AAV3 vectors were analyzed by qPCR using transgene-specific primers for presence of the AAV genome.

[0136] Groups of 20 C57Bl / 6 and 20 C3-KO mice were injected either once with 2 x 1011vg or 4 times in 2-weekly intervals with 5 x 1010vg of the of the AAV3-VP2- GFP vector. In the single high dose group 4 mice were euthanized 7 days, 4 weeks, 3, 6 or 12 months later. In the 4x low dose vector group where Abs induced by the 1stdose of the AAV vector may contribute to the deposition of AAV on FDCs mice were euthanized 1 week after the 1stdose, 1 week after the 2ndand 3rddose, and 3, 6 or 12 months after the last dose. Lymph nodes (inguinal, popliteal, cervical, periaortal, axillary and brancial) were harvested, cut into pieces, and treated with 100 μg / ml DNase I, 800 μg / ml dispase, and 200 μg / ml collagenase. Cells were filtered through a mesh and single cell suspensions will be treated with magnetic beads coated with anti-CD3 and anti-CD45 to remove B cells and T cells. Cells were then treated with fluorochrome labeled Abs to FDC markers, i.e., Ter119, CD31, Podoplanin, CD35, CD21 and CD23. Fluorochromes that lack spectral overlap with GFP were selected. Upon flow cytometry cells were gatedon Ter119-CD31-Podoplanin+CD35+CD21+CD23+FDCs and the percentage of GFP+cells within this population were determined. Two lymph nodes from each mouse were set aside for immunohistochemical analyses. Lymph nodes embedded in OCT were snap frozened and 10 µM sections upon rehydration and blocking were stained with an PE- labeled Ab to MFG-E8 also known as FDC-M1, an APC-labeled anti-CD169 Ab that stains marginal zone macrophages and thus visualizes GCs and B220 to stain B cells. Sections were analyzed by confocal microscopy for GFP+(FITC+)FDC-M1+cells as well as for GFP+(FITC+)FDC-M1-B220+B cells to determine if C3 affects numbers and persistence of AAV particles in lymph nodes. Immunogenicity of AAV vectors with a capsid selected for lack of iC3b binding

[0137] Binding sites between AAV capsid and iC3b were unknown although the model in Figure 5 suggests that the capsid’s HI loop may be involved. To substantiate this model, an iC3b deletion mutant was used and then cryo-EM of AAV3-iC3b complexes, which allows for identification of iC3b binding residues on AAV capsid. This in turn enabled us to develop a modified AAV3 capsid that no longer binds iC3b, which was expected to markedly reduce its ability to induce Ab responses. It should be noted that mutation of the HI loop or other sequences may change not only VP3 but also VP1 and VP2 as all 3 are encoded by one open reading frame. Previous experiments have shown that point mutations in all three proteins are tolerated by AAV, whose capsid is composed of a 1:1:10 ratio of VP1, VP2and VP3.

[0138] Complex formation binding of iC3b to highly purified AAV3 particles was ensured and tested using an ELISA in which AAV particles are coated to plastic wells and the treated with 10-200 excess of iC3b molecules to determine binding saturation. To validate the model shown in Figure 5 a full length human iC3b with an N- terminal HA-tag was generated. A mutant form of iC3b from which amino acids 243 to 538, which form the putative binding site to AAV capsid have been removed, was constructed (Figure 5). The mutant form was also linked to an HA tag. The proteins were expressed in CHO cells and purified from culture supernatant using a commercially available HA-tagged protein purification kit. Concentration of purified protein was determined by a BCA protein assay. Equal amounts of the full-length and mutant iC3bwas then tested in an ELISA on plates coated with AAV3 using an anti-HA Ab for detection of bound iC3b.

[0139] For the cryo-EM studies, a ratio of iC3b to AAV particles (10-20 µl of empty particles at 3 x 1013vg equivalent / ml) was used that is approximately double of that resulting in saturated binding. AAV3 / iC3b complex were purified using gel filtration, applied to cryo-EM grids, and vitrified with a Vitrobot Mark IV (FEI Co.). The grids were screened and imaged with a Titan Krios G3i cryogenic transmission electron microscope, which allows for high resolutions beyond 3Å.

[0140] Libraries of AAV variants with multiple distinct mutations within their receptor binding domain or binding sites for neutralizing Abs were generated based on structural data from cryo-EM images. Data on the iC3b binding site was used to generate mutations within the binding site. High resolution images were achieved from cryo-EM allowing for identification of interacting residues which were employed for structure- guided design to knock out the binding site. AAV3 / iC3b structure was used as input in a Rosetta design protocol (Pallesen J, et al., Proc Natl Acad Sci U S A.2017 Aug 29;114(35):E7348-E7357) wherein key residues were mutated to abrogate the interaction and computationally down-select capsid mutations to identify promising candidates. The resolution allowed for identification of an area where iC3b binds but not the individual amino acids. AAV libraries with alternate amino acids of these residues were then created. These AAV variant libraries consisted of AAV variant capsids with the simultaneous substitution of the iC3b binding natural amino acids with any of the 20 amino acids (5-7 individual amino acid of iC3b binding AAV residues). These capsid variants replaced the wild-type AAV3b capsid gene in a replication competent AAV genome that also expresses AAV2-Rep. AAV3b mutant libraries were produced by transfection of HEK293 cells with these plasmids together with a helper plasmid providing the adenoviral helper functions. The complexity of the AAV libraries were determined by Illumina or PacBio NGS. To negatively select for iC3b binding, tosyl- activated dynabeads were coated with iC3b at 4oC overnight following the manufacturer’s instructions. After coupling, the bead surface was rendered inert to prevent further binding. Beads were then incubated at room temperature for 2 hours with the AAV library and removed by a magnet. This was repeated twice. The remaininglibrary was expanded. An aliquot was used to expand the cap sequence by PCR and 20 clones were sequenced to assess loss of diversity of the capsid sequence. Other aliquots of the library were injected via tail vein into mice. Three days later livers were isolated and viral capsid DNA were extracted and amplified by PCR. The pooled PCR product were cloned into a plasmid containing cap to generated replication competent AAV in HEK293 cells that are transfected with a helper plasmid. The selection procedure was repeated and then 20 clones of the PCR amplicon was sequenced to detect clones that became dominant after the selection procedure. Sequences encoding the capsid of the 2-3 most dominant clones were amplified by PCR and cloned into pSSV9, which were used to generate iC3b-modified AAV3 (AAV3iC3bdel) vectors expressing human F.IX. The vectors were tested in mice upon i.v. injection in comparison to a wild-type AAV3 vector for induction of neutralizing Abs and hF.IX levels.

[0141] The experiments described above was repeated with the two most efficacious drug treatments with this AAViC3bdel vector and a wild-type vector.

[0142] Presumably the iC3b binding site within the capsid antigens is conserved between most AAV serotypes. Once the site within the capsid of AAV3 was identified and shown that its modification ablated iC3b binding reducing and shortening AAV capsid-specific Ab responses, the sequence of this site was compared with different commonly used AAV serotypes, and the mutation was incorporated into additional capsid(s). Some of the biological assays was then repeated with the additional modified serotype to confirm that such a mutation has general value to reduce B cell responses to AAV-mediated gene transfer. Significance

[0143] AAV-mediated gene transfer has tremendous potential to cure monogenic disorders. Although gene transfer with moderate doses of AAV vectors is well tolerated the very high doses that are needed to correct many diseases have led to SAEs and deaths even in patients that were concomitantly treated with immunosuppressive drugs. One potential approach to allow for high doses of AAV vectors without unacceptable risk would be the repeated transfer of lower vector doses, but again this is problematic as AAV vectors induce very potent capsid-specific neutralizing Ab responses that precludethe success of additional gene transfer using a serologically cross-reactive AAV vector. Several immunosuppressive regimens have been tried in experimental animals and although they reduced Ab responses to AAV capsid the effect was in general transient, and Abs rebounded once drug treatment was stopped presumably reflecting presence of residual AAV capsid antigen with germinal centers. This invites the exploration of additional drug combinations that target different pathways of B cell activation. Drugs by themselves may not suffice to inhibit B cell responses to AAV capsid. We hypothesize that the exceptionally high immunogenicity of AAV capsid is in part driven by its ability to bind iC3b which augments antigen deposition on FDCs and upon binding the antigen to CD19 amplifies B cell receptor signaling. Removing the iC3b binding site from AAV capsid to expected to markedly reduce and shorten capsid specific B cell responses and such lowered responses are likely to be more amendable to drug-mediated inhibition.

[0144] Such mutated AAV vectors combined with drug treatment would remove the main obstacle for the repeated use of AAV vectors for gene transfer.

[0145] A systematic study of drugs that inhibit molecules of key signaling pathways of B cell on antibody responses to repeated AAV gene transfer has not yet been conducted.

[0146] Although some studies have explored the role of complement C3 fragments on innate or adaptive immune responses to AAV vectors these studies did not analyze their effects on susceptibility of B cell activation to immunoinhibitory drugs.

[0147] In addition, it has not yet been explored how C3 fragments affect antigen deposition and longevity on FDCs, which is likely a key parameter that drive the very sustained Ab responses to AAV and caused breakthrough Ab responses to AAV capsid in immunosuppressed mice once drug treatment is stopped.

[0148] Identifying the residues on AAV capsid that bind to iC3b through cryo- EM studies will allow targeted destruction of this binding site either through point mutations or the generation of an AAV library followed by selection of loss of iC3b mutants. Such mutants if they induce lower and shorted Ab responses as will be tested would be exceeding valuable for gene transfer.

[0149] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A composition comprising an engineered adeno-associated virus (AAV) virion, wherein the AAV capsid comprises one or more amino acid mutations that inhibits its binding to C3b.

2. The composition of claim 1, wherein the AAV is AAV3.

3. The composition of claim 1, comprising a nucleic acid molecule comprising a heterologous nucleotide sequence encoding a therapeutic agent flanked by AAV ITRs.

4. The composition of claim 3, wherein the heterologous nucleotide sequence is inserted into the genome of a target cell.

5. A composition comprising a nucleic acid molecule, or combination thereof, comprising the set of genes for generation of the AAV virion of any one of claims 1 to 4.

6. The composition of claim 5, comprising a nucleotide sequence encoding an AAV capsid comprising one or more amino acid mutations that inhibits its binding to C3b and further comprising at least one transgene flanked by AAV ITRs.

7. A method of gene therapy comprising administering a composition of any one of claims 1 to 6 to a subject in need thereof.

8. The method of claim 7, wherein the composition is administered more than once.

9. The method of claim 7, wherein the composition is administered in combination with at least one additional agent.

10. The method of claim 9, wherein the composition is administered in combination with a helper virus or helper vector.

11. The method of claim 9, wherein the composition is administered in combination with an immunosuppressive agent.

12. The method of claim 9, wherein the composition is administered in combination with a B cell inhibitor.

13. The method of claim 9, wherein the composition is administered in combination with a C3 complement fragment, Fostamatinib, Ibrutinib, Idelalisib or Bortezomib.

14. The method of claim 7, wherein the gene therapy is administered for the treatment of Duchenne muscular dystrophy (DMD), thalassemia, sickle cell disease, cystic fibrosis, polycystic kidney disease, or Tay–Sachs disease.

15. A method of reducing or preventing the generation of neutralizing antibodies (NAs) in a subject, the method comprising administering at least one B-cell inhibitor.

16. The method of claim 15, wherein at least one B-cell inhibitor is administered in combination with at least one AAV vector.

17. The method of claim 15, wherein at least one B-cell inhibitor is administered in combination with the composition of any one of claims 1 to 6.

18. The method of any one of claims 15 to 17, wherein the B-cell inhibitor is a C3 complement fragment, Fostamatinib, Ibrutinib, Idelalisib or Bortezomib.

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