Screening cell-type-specific AAV capsids

Abstract

Aspects of the disclosure relate to compositions and methods for expressing adeno- associated virus capsid proteins. The disclosure relates, in part, to novel synthetic AAV vectors comprising a Rep gene positioned upstream of a 5' inverted terminal repeat (ITR), a promoter operably linked to a capsid protein-encoding nucleic acid sequence. Methods of producing an rAAV capsid library are also disclosed.

Description

[0001] SCREENING CELL-TYPE-SPECIFIC AAV CAPSIDS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. provisional Application Serial Number 63 / 596,610, filed November 6, 2023, entitled “SCREENING CELL-TYPE-SPECIFIC AAV CAPSIDS,” the entire contents of which are incorporated by reference herein.

[0004] FIELD OF THE DISCLOSURE

[0005] The disclosure in some aspects relates to isolated nucleic acids, compositions, and kits useful for identifying adeno-associated viruses in cells. In some aspects, the disclosure provides novel AAVs and methods of use thereof as well as related kits.

[0006] BACKGROUND OF INVENTION

[0007] Adeno-associated virus (AAV) vector is a key platform technology for gene therapy applications (e.g., for the treatment of human diseases). An AAV vector is typically comprised of two viral genes - rep (replication) and cap (capsid), encoding non- structural and structural proteins, respectively. The rep and cap genes are typically flanked by 145 nucleotide-long inverted terminal repeats (ITR) at the terminal ends of the genome. The capsid protein, which assists with transport and delivery of the viral genome to host cells, can be designed to preferentially target certain cell types. To advance capsid design and development, AAV libraries have been a powerful tool for capsid engineering and yielded many promising capsids for in vivo gene transfer. An ideal approach for AAV library preparation should be capable of efficiently generating highly diverse libraries with minimal cross-packaged genome and mosaicism of capsid. Importantly, a function-based library screening is desired to select cell- type-specific capsids.

[0008] SUMMARY OF INVENTION

[0009] Aspects of the disclosure relate to methods for the screening and identification of new AAV capsid proteins (e.g., cell-type-specific AAV capsid proteins). The disclosure is based, in part, on generation of a recombinant AAV genome in which the Rep gene is positioned at the upstream of a 5' inverted terminal repeat (ITR) to drive Cap gene expression during vector manufacturing (see, “Recombinant genome” of FIG. 1A). An AAV capsid library can be generated in which each wildtype AAV-like (“WT AAV-like”) vector of the library encodes a unique AAV capsid protein. In some embodiments, expression of the AAV capsids within the library is driven by a constitutive or cell-type-specific promoter, thereby enabling in vivo selection based on AAV capsid expression in a desired tissue. This disclosure allows for the use of tissue-specific promoters without AAV Rep sequence to drive capsid mRNA for selection in vivo, by incorporating the Rep gene, ITR, and Cap gene (encoding the capsid) in the same plasmid. This methodology produces a capsid library with minimal library plasmid input while also reducing vector genome cross-packaging and capsid mosaicism.

[0010] Accordingly, in some aspects, the disclosure provides a nucleic acid (e.g., a WT AAV- like vector) comprising, in a 5' to 3' orientation, a Rep gene, a 5' inverted terminal repeat (ITR), a promoter, a Cap gene, and a 3' ITR.

[0011] In some embodiments, the nucleic acid does not comprise an ITR located upstream of the Rep gene (e.g., located at the 5' end of the Rep gene).

[0012] In some embodiments, the promoter is a constitutive promoter or a tissue- specific promoter. In some embodiments, the promoter is a cTnT promoter, a chicken beta actin (CBA) promoter, a U1 a promoter, a MAGI promoter, a Syn promoter, a MECP2 promoter, a CD68 promoter, a MCK7 promoter, a CK8e promoter, a TBG promoter, a Gfap promoter, or a SMN1 promoter.

[0013] In some embodiments, the 5’ ITR or the 3’ ITR is an AAV2 ITR. In some embodiments, the 5’ ITR or the 3’ ITR is a mutant ITR.

[0014] In some embodiments, the Rep gene encodes one or more AAV viral replication proteins. In some embodiments, the one or more AAV viral replication proteins are selected from Rep78, Rep68, Rep52 and Rep40. In some embodiments, the Cap gene encodes an AAV capsid protein.

[0015] Some aspects of the disclosure provide a method of producing a capsid protein, the method comprising transfecting a host cell with a nucleic acid as described herein.

[0016] Some aspects of the disclosure provide a method of producing a capsid protein, the method comprising administering a nucleic acid as described herein to a subject.

[0017] In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human, a mouse, a rat, a monkey, or a pig.

[0018] Further aspects of the disclosure provide an AAV capsid library comprising a plurality of unique WT AAV-like vectors; wherein each unique WT AAV-like vector comprises, in a 5' to 3' orientation, a Rep gene, a 5' inverted terminal repeat (ITR), a promoter, a Cap gene, and a 3' ITR; and wherein the Cap gene of each WT AAV-like vector encodes a unique AAV capsid protein, relative to the other WT AAV-like vectors of the plurality.

[0019] In some embodiments, the promoter is a constitutive promoter or a tissue- specific promoter. In some embodiments, each WT AAV-like vector of the library comprises a promoter independently selected from: a cTnT promoter, a chicken beta actin (CBA) promoter, a Ula promoter, a MAGI promoter, a Syn promoter, a MECP2 promoter, a CD68 promoter, a MCK7 promoter, a CK8e promoter, a TBG promoter, a Gfap promoter, or a SMN1 promoter. In some embodiments, the promoter is a cTnT promoter, a chicken beta actin (CBA) promoter, a Ula promoter, a MAGI promoter, a Syn promoter, a MECP2 promoter, a CD68 promoter, a MCK7 promoter, a CK8e promoter, a TBG promoter, a Gfap promoter, or a SMN1 promoter.

[0020] In some embodiments, the AAV capsid library comprises 10-1000 unique WT AAV-like vectors, each unique WT AAV-like vector comprising a unique AAV capsid protein.

[0021] In some embodiments, the unique AAV capsid protein of each WT AAV-like vector has at least one amino acid insertion, substitution, or deletion relative to at least one other unique AAV capsid protein of the library.

[0022] In some embodiments, the WT AAV-like vector does not have an ITR that is located upstream of the Rep gene (e.g., located at the 5' end of the Rep gene).

[0023] In some embodiments, the Rep gene encodes one or more AAV viral replication proteins. In some embodiments, the one or more AAV viral replication proteins are selected from Rep78, Rep68, Rep52 and Rep40.

[0024] Further aspects of the disclosure provide a method of screening WT AAV-like vectors in vivo, the method comprising administering an AAV capsid library described herein to one or more subjects.

[0025] In some embodiments, the one or more subjects are one or more mammalian subjects. In some embodiments, the one or more subjects are one or more humans, mice, rats, monkeys, or pigs.

[0026] In some embodiments, the unique AAV capsid protein of each of the unique WT AV-like vectors are preferentially expressed in a target tissue of the subject.

[0027] In some embodiments, the method further comprises isolating the unique capsid proteins from the subject. In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) particle comprising: a recombinant adeno-associated virus (rAAV) vector; and a capsid protein isolated from a subject administered a capsid library as described herein.

[0028] BRIEF DESCRIPTION OF DRAWINGS

[0029] FIG. 1A shows the design of AAV vectors of the disclosure comprising in a 5' to 3' orientation, a Rep gene, a 5' inverted terminal repeat (ITR), a promoter, a Cap gene, and a 3' ITR (“Recombinant Genome”). A library of AAV capsids can be generated using these AAV vectors by designing unique AAV capsid proteins using domain swapping, peptide insertion, natural variant mutations, and in silica synthesis.

[0030] FIG. IB shows that the AAV vectors of the disclosure can be produced in high yields.

[0031] FIG. 2 shows that an AAV vector of the disclosure can successfully package into its capsid protein when transfected in the presence of a traditional cis-AAV plasmid.

[0032] FIGs. 3A-3D show representative data for AAV yield using methods described by the disclosure. FIG. 3A shows a schematic depicting the wildtype (WT) AAV genome (top) and a WT AAV-like genome (bottom). FIG. 3B shows the vector yield of WT AAV-like by transfection with 150, 50, 17 and 5.0 pg plasmids and 150 pg adeno-helper plasmid and tripletransfection with 150 pg adeno-helper, 150 pg Rep2-Cap9, and different amount of ITR plasmid carrying the same vector genome but no Rep gene outside of ITR. FIG. 3C shows genome composition of AAV vectors produced from WT AAV-like plasmids containing AAV9, AAV9- null which the start codon of VP1, VP2 and VP3 are mutated into stop codon, or the mixture of these two plasmids at different ratio. The ratios of AAV9 and AAV9-null were calculated using band intensities. The genomic DNA was extracted from purified vectors for PCR to amply the fragment that span the C-terminal of capsid gene and polyA. The PCR fragment from AAV9 and AAV9-null can be digested by Pmel and Nhel, respectively. The ratios of AAV9 and AAV9-null were calculated using band intensities. FIG. 3D shows genome composition of AAV vectors produced from WT AAV-like plasmids containing AAV8, AAV9, or the mixture of these two plasmids at different ratio. The ratios of AAV8 and AAV9 were calculated using band intensities. The genomic DNA was extracted from purified vectors for PCR to amply the fragment that span the N-terminal of capsid genes. The PCR fragment from AAV8, but not AAV9 can be digested by Agel. The ratios of AAV8 and AAV9 were calculated using band intensities. DETAILED DESCRIPTION

[0033] Aspects of the disclosure relate to compositions and methods for producing viral vectors and viral particle, for example recombinant adeno-associated virus (rAAV) vectors and particles. The disclosure is based, in part, on wildtype AAV-like (WT AAV-like) vectors comprising a nucleic acid sequence encoding a Rep protein positioned outside of the 5’ inverted terminal repeat (ITR) of a recombinant AAV vector genome encoding a capsid protein. In some embodiments, the WT AAV-like vectors of the disclosure are useful for encoding capsid proteins that are components of a capsid protein library, which may be screened to identify capsid proteins having desirable characteristics, e.g., tropism for a target tissue. In some embodiments, WT AAV-like vectors of the disclosure reduce the number of cross-packaged or capsid mosaics present in the capsid library. Aspects of the disclosure relate to methods for screening capsid libraries produced using WT AAV-like vectors described herein.

[0034] WT AAV-like vectors

[0035] Aspects of the disclosure relate to nucleic acids encoding a wildtype AAV-like (WT AAV-like) vector. In some embodiments, a WT AAV-like vector comprises a nucleic acid sequence encoding a Rep protein positioned outside of the 5’ inverted terminal repeat (ITR) of a recombinant AAV vector genome encoding a capsid protein. In some embodiments, the rAAV vector genome encoding the capsid protein comprises a 3’ ITR positioned downstream (e.g., 3’ relative to, for example after the last codon) of the capsid protein encoding nucleotide sequence.

[0036] A "nucleic acid" sequence refers to a DNA or RNA sequence. In some embodiments, the term nucleic acid captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxy hydroxyl-methyl) uracil, 5-fluorouracil, 5- bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudo-uracil, 1- methylguanine, 1 -methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3- methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- oxyacetic acid methylester, uracil-5-oxy acetic acid, oxybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6- diaminopurine.

[0037] In some embodiments, proteins and nucleic acids of the disclosure are isolated. As used herein, the term “isolated” means artificially obtained or produced. As used herein with respect to nucleic acids, the term “isolated” generally means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one that is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins or peptides, the term “isolated” generally refers to a protein or peptide that has been artificially obtained or produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0038] It should be appreciated that conservative amino acid substitutions may be made to provide functionally equivalent variants, or homologs of the capsid proteins. In some aspects the disclosure embraces sequence alterations that result in conservative amino acid substitutions. As used herein, a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, one can make conservative amino acid substitutions to the amino acid sequence of the proteins and polypeptides disclosed herein. In some embodiments, a WT AAV-like vector comprises a nucleic acid sequence encoding a Rep protein positioned upstream of a recombinant AAV vector. As used herein, “Recombinant AAV (rAAV) vectors” of the disclosure are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence. In some embodiments, the nucleic acid sequence is heterologous to the vector sequences, and encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. In some embodiments, a transgene encodes a capsid protein, for example an adeno-associated virus (AAV) capsid protein. The nucleic acid coding sequence is operatively linked to regulatory components in a manner that permits transgene transcription, translation, and / or expression in a cell of a target tissue.

[0039] The AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present disclosure is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the AAV ITRs are AAV2 ITRs.

[0040] In some embodiments, the disclosure provides a self-complementary AAV vector. As used herein, the term “self-complementary AAV vector” (scAAV) refers to a vector containing a double-stranded vector genome generated by the absence of a terminal resolution site (TR) from one of the ITRs of the AAV. The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present. In general, scAAV vectors generate singlestranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle. In some embodiments, the rAAVs of the present disclosure are pseudotyped rAAVs. Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle. With this method, the foreign viral envelope proteins can be used to alter host tropism or an increased / decreased stability of the virus particles. In some aspects, a pseudotyped rAAV comprises nucleic acids from two or more different AAVs, wherein the nucleic acid from one AAV encodes a capsid protein and the nucleic acid of at least one other AAV encodes other viral proteins and / or the viral genome. In some embodiments, a pseudotyped rAAV refers to an AAV comprising an inverted terminal repeat (ITR) of one AAV serotype and a capsid protein of a different AAV serotype (or a variant of a capsid of the same type as the ITR). For example, a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX / Y (e.g., AAV2 / 1 has the ITRs of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAVs may be useful for combining the tissue- specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue.

[0041] In addition to the major elements identified above for the recombinant AAV vector, the rAAV vector also includes conventional control elements necessary which are operably linked to the transgene 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 disclosure. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0042] Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters that are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0043] As used herein, a nucleic acid sequence (e.g., coding sequence) and regulatory sequences are said to be “operably” linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5’ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame. In some embodiments, operably linked coding sequences yield a fusion protein. In some embodiments, operably linked coding sequences yield a functional RNA e.g., shRNA, miRNA, miRNA inhibitor).

[0044] For nucleic acids encoding proteins, a poly adenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence. A rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence would be used to produce a protein that contains more than one polypeptide chains. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al, and references cited therein at, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, a Foot and Mouth Disease Virus 2A sequence is included in polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

[0045] The precise nature of the regulatory sequences needed for gene expression in host cells may vary between species, tissues or cell types, but shall in general include, as necessary, 5’ non-transcribed and 5’ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like. Especially, such 5’ non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the disclosure may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

[0046] In some embodiments, a WT AAV-like vector comprises a promoter operably linked to the nucleic acid sequence encoding a capsid protein. In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen].

[0047] In some embodiments, the promoter of a WT AAV-like vector is a constitutive promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc -inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline- repressible system (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and the rapamycin- inducible system (Magari et al, J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0048] In another embodiment, the native promoter for the transgene will be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue- specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0049] A promoter of a WT AAV-like vector may be a ubiquitous promoter (e.g., functional in many or all cells / tissues of a subject) or a tissue-specific promoter. In some cases, the tissuespecific regulatory sequences (e.g., promoters) bind tissue- specific transcription factors that induce transcription in a tissue specific manner. Such tissue- specific regulatory sequences e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue- specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, a gastrointestinal- specific mucin-2 promoter, an eye- specific retinoschisin promoter, an eye-specific K12 promoter, a respiratory tissue-specific CC10 promoter, a respiratory tissue-specific surfactant protein C (SP-C) promoter, a breast tissuespecific PRC1 promoter, a breast tissue- specific RRM2 promoter, a urinary tract tissue-specific uroplakin 2 (UPII) promoter, a uterine tissue- specific lactoferrin promoter, or a cardiac Troponin T (cTnT) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal such as neuron- specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron- specific vgf gene promoter (Piccioli et al., Neuron, 15:373- 84 (1995)), among others which will be apparent to the skilled artisan.

[0050] In some embodiments, a WT AAV-like vector comprises a promoter selected from a cTnT promoter, a chicken beta actin (CBA) promoter, a Ula promoter, a MAGI promoter, a Syn promoter, a MECP2 promoter, a CD68 promoter, a MCK7 promoter, a CK8e promoter, a TBG promoter, a Gfap promoter, and a SMN1 promoter.

[0051] Isolated Capsid Proteins

[0052] Aspects of the disclosure relate to nucleic acids (e.g., WT AAV-like vectors) encoding one or more capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by a cap gene of an AAV. In some embodiments, AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which may be expressed from a single cap gene. Accordingly, in some embodiments, the VP1, VP2 and VP3 proteins share a common core sequence. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the protein shell is primarily comprised of a VP3 capsid protein. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner. In some embodiments, VP1 and / or VP2 capsid proteins may contribute to the tissue tropism of the packaged AAV. In some embodiments, the tissue tropism of the packaged AAV is determined by the VP3 capsid protein. In some embodiments, the tissue tropism of an AAV is enhanced or changed by mutations occurring in the capsid proteins.

[0053] AAV capsid proteins are well known, for example as described by Wang et al. Signal Transduction and Targeted Therapy, volume 9, Article number: 78 (2024). In some embodiments, AAV capsid proteins are wildtype AAV capsid proteins (e.g., capsid proteins isolated from naturally occurring AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.74, etc.). In some embodiments, AAV capsid proteins are naturally occurring variants of wildtype AAV capsid proteins, for example capsid proteins having amino acid sequences having one or more substitutions, insertions, or deletions relative to a wildtype AAV capsid protein sequence. In some embodiments, AAV capsid proteins are engineered AAV capsid proteins (e.g., AAV capsid proteins comprising one or more amino acid sequences that are not naturally occurring in AAV capsid proteins, for example peptide-grafted AAV capsid proteins). In some embodiments, AAV capsid proteins are chimeric AAV capsid proteins (e.g., AAV capsid proteins comprising a first region derived from a first AAV capsid protein and a second region derived from a second AAV capsid protein).

[0054] The disclosure provides in some aspects novel AAV capsid proteins that are encoded by WT AAV-like vectors described herein. In some embodiments, the AAV capsid proteins comprise one or more amino acid substitutions, insertions, or deletions relative to a wildtype AAV capsid protein. In some embodiments, an AAV capsid protein encoded by a WT AAV-like vector of the disclosure comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90% homologous to a wildtype AAV capsid protein. In some embodiments, capsid proteins encoded by WT AAV-like vectors described herein specifically target various in vivo tissues e.g., liver, brain, gastric, respiratory, breast, pancreatic, rectal, prostate, urologic, and cervical tissues).

[0055] “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. The term "substantial homology", when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in about 90 to 100% of the aligned sequences. When referring to a polypeptide, or fragment thereof, the term “substantial homology” indicates that, when optimally aligned with appropriate gaps, insertions or deletions with another polypeptide, there is nucleotide sequence identity in about 90 to 100% of the aligned sequences. The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. In some cases, highly conserved may refer to 100% identity. Identity is readily determined by one of skill in the art by, for example, the use of algorithms and computer programs known by those of skill in the art.

[0056] As described herein, alignments between sequences of nucleic acids or polypeptides are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs, such as "Clustal W", accessible through Web Servers on the internet. Alternatively, Vector NTI utilities may also be used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using BLASTN, which provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Similar programs are available for the comparison of amino acid sequences, e.g., the "Clustal X" program, BLASTP. Typically, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. Alignments may be used to identify corresponding amino acids between two proteins or peptides. A “corresponding amino acid” is an amino acid of a protein or peptide sequence that has been aligned with an amino acid of another protein or peptide sequence. Corresponding amino acids may be identical or non-identical. A corresponding amino acid that is a non-identical amino acid may be referred to as a variant amino acid.

[0057] Alternatively for nucleic acids, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single- stranded- specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

[0058] Methods for Discovering AAVs

[0059] Much of the biology of AAV is influenced by its capsid protein. Consequently, methods for discovering novel AAVs have been largely focused on isolating DNA sequences for AAV capsids. A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of integrated and / or episomal genomes in a host cell. Methods used for isolating novel AAV include PCR based molecular rescue of latent AAV DNA genomes, infectious virus rescue of latent proviral genome from tissue DNAs in vitro in the presence of adenovirus helper function, and rescue of circular proviral genome from tissue DNAs by rolling-circle-linear amplification, mediated by an isothermal phage Phi-29 polymerase. All of these isolation methods take advantage of the latency of AAV proviral DNA genomes and focus on rescuing persistent viral genomic DNA.

[0060] In some aspects, the disclosure relates to the discovery that novel AAV variants with desirable tissue tropisms can be identified from in vivo tissues of a subject. Without wishing to be bound by any particular theory, the use of in vivo tissue exploits the natural reservoir of genomic diversity observed among viral genomic sequences isolated from both normal and tumor tissues of a subject. Thus, in some embodiments, in vivo tissues act as natural incubators for viral (e.g., viral capsid protein) diversity through selective pressure and / or immune evasion.

[0061] In some aspects, the disclosure relates to the discovery that viral particles produced by infecting a subject with a library of WT AAV-like vectors described by the disclosure can be used to identify novel capsid protein variants. Without wishing to be bound by any particular theory, the use of WT AAV-like vectors described herein results in reduced cross-packaging and capsid mosaicism and improves capsid library fidelity relative to capsid variants obtained from other conventional methodologies such as TRACER (Tropism Redirection of AAV by Cell- type-specific Expression of RNA).

[0062] As used herein, “cross-packaging” refers to an AAV genome being packaged into a mismatched capsid. In some embodiments, administering a capsid protein library comprising a plurality of WT AAV-like vectors as described herein to a subject reduces the instances of crosspackaging in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to previously described AAV capsid library screening methods.

[0063] As used herein, “capsid mosaicism” refers to oligomerization of distinct variant VP 1-3 proteins into a single viral particle. In some embodiments, administering a capsid protein library comprising a plurality of WT AAV-like vectors as described herein to a subject reduces the instances of capsid mosaicism in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to previously described AAV capsid library screening methods.

[0064] A library of WT AAV-like vectors may comprise any number of unique capsid sequences. In some embodiments, a library of WT AAV-like vectors encodes at least 10, 100, 200, 500, 750, 1000, or more unique capsid proteins. In some embodiments, a library of WT AAV-like vectors encodes between 10 and 1000 unique capsid proteins. “Unique” capsid proteins refers to capsid protein sequences that differ from one another by at least one amino acid (e.g., no two capsid proteins of the library comprise an amino acid sequence having exactly the same amino acid composition and / or length).

[0065] Aspects of the disclosure relate to methods for screening capsid proteins. In some embodiments, the methods comprise administering a capsid protein library (e.g., a plurality of WT AAV-like vectors, each vector encoding a unique capsid protein) to a subject. In some embodiments, the capsid proteins or viral particles produced in the subject are subsequently isolated. Thus, capsid proteins having novel properties may be identified. In some embodiments, novel capsid proteins of the disclosure may be identified by isolating cap mRNA from the transfected cells, creating cDNA from the mRNA (e.g., by RT-PCR) and sequencing the cDNA.

[0066] In some aspects, the disclosure provides isolated AAVs. As used herein with respect to AAVs, the term “isolated” refers to an AAV that has been artificially obtained or produced. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs.” Recombinant AAVs (rAAVs) preferably have tissue- specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s). The AAV capsid is an important element in determining these tissuespecific targeting capabilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected. Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003 / 0138772), the contents of which are incorporated herein by reference in their entirety).

[0067] The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0068] The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector).

[0069] As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment (e.g., nucleic acid sequence) to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) from a transcribed gene.

[0070] In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell (e.g., across the cell membrane). A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

[0071] A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell that has been transfected. Thus, a “host cell” as used herein may refer to a cell that 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.

[0072] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0073] As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

[0074] Cells may also be transfected with a vector (e.g., helper vector) that provides helper functions to the AAV. The vector providing helper functions may provide adenovirus functions, including, e.g., Ela, Elb, E2a, and E4ORF6. The sequences of adenovirus gene providing these functions may be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified human types known in the art. Thus, in some embodiments, the methods involve transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0075] In some cases, an isolated capsid gene can be used to construct and package recombinant AAVs, using methods well known in the art, to determine functional characteristics associated with the capsid protein encoded by the gene. For example, isolated capsid genes can be used to construct and package a recombinant AAV (rAAV) comprising a reporter gene (e.g., B- Galactosidase, GFP, Luciferase, etc.). The rAAV can then be delivered to an animal (e.g., mouse) and the tissue targeting properties of the novel isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues (e.g., heart, liver, kidneys) of the animal. Other methods for characterizing the novel isolated capsid genes are disclosed herein and still others are well known in the art.

[0076] The foregoing methods for packaging recombinant vectors in desired AAV capsids to produce the rAAVs of the disclosure are not meant to be limiting and other suitable methods will be apparent to the skilled artisan.

[0077] The WT AAV-like vectors (e.g., rAAV particles each comprising a WT AAV-like vector) may be delivered to a subject in compositions according to any appropriate methods known in the art. The rAAVs, preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a nonhuman primate (e.g., Macaque). In some embodiments a host animal does not include a human.

[0078] Delivery of the WT AAV-like vectors (e.g., rAAV particles each comprising a WT AAV-like vector) to a mammalian subject may be by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAVs. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, in certain instances, it may be desirable to deliver the 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, cereobrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like. Recombinant AAVs 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 (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAVs (e.g., rAAV particles each comprising a WT AAV-like vector) is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

[0079] Optionally, the compositions of the disclosure may contain, in addition to the rAAVs 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.

[0080] The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.

[0081] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.

[0082] An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary between animals or tissues. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109to 1016genome copies. In some embodiments the rAAV is administered at a dose of IO10, IO11, 1012, IO13, 1014, or 1015genome copies per subject. In some embodiments the rAAV is administered at a dose of IO10, 1011, 1012, 1013, or 1014genome copies per kg. In some cases, a dosage between about 1011to 1012rAAV genome copies is appropriate. In certain embodiments, 1012rAAV genome copies is effective to target heart, liver, and pancreas tissues. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

[0083] In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ~1013GC / ml or more). Methods for reducing aggregation of rAAVs are well-known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)

[0084] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

[0085] Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0086] In certain circumstances it will be desirable to deliver the rAAVs (e.g., rAAV particles each comprising a WT AAV-like vector) in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver rAAVs. In some embodiments, a preferred mode of administration is by portal vein injection. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0087] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0088] Sterile injectable solutions are prepared by incorporating the active rAAVs in the required amount in the appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0089] The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0090] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0091] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.

[0092] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAVs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587). Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

[0093] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500. ANG., containing an aqueous solution in the core.

[0094] Alternatively, nanocapsule formulations of the rAAVs may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0095] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAVs to a host. Sonophoresis (z.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).

[0096] EXAMPLES

[0097] Example 1. Development of an expression based AAV capsid selection platform.

[0098] An expression-based AAV capsid selection platform was developed to enable the screening of cell-type-specific AAV capsids. The Rep gene was located at the upstream of the 5' inverted terminal repeat (ITR) to drive the Cap gene expression during vector manufacturing (see, “Recombinant genome” of FIG. 1A). This contrasts with the wild-type (WT) AAV genome (see, FIG. 1A) in which the 5' ITR is located upstream of the Rep gene. The AAV capsid library was driven by a constitutive or cell-type- specific promoter, enabling in vivo selection based on the capsid mRNA expression (FIG. 1A).

[0099] The platform showed significant improvement in the vector yield. Using the same amount of plasmid as conventional triple plasmid transfection, the vector yield increases 5-10- fold. With 3.3% of the platform plasmid, the vector yield is comparable to conventional approaches (FIG. IB).

[0100] To test if the platform could reduce the cross-packaged genomes, regular cis-AAV plasmid (#1) and platform plasmid (#2) were mixed at the same mass ratio for vector production. The vector genomic DNA was extracted from purified vectors and genome composition was examined on denatured agarose gel. The analysis showed that AAV capsid sequences tend to be packaged into its corresponding capsid when lower amounts of plasmid are used for vector production (FIG. 2).

[0101] Example 2. Wildtype AAV -like (WT AAV-like) capsid screening platform.

[0102] AAV9 can cross the blood-brain barrier (BBB) following systemic delivery, but most vectors transduce non-CNS tissues. New AAV capsids with more efficient transduction should result in better therapeutic efficacy while lowering the high-dose vector-related safety concerns and manufacturing costs. Pooled and multiplex AAV libraries emerged as a powerful tool for capsid engineering with improved properties. ITR-containing rep / cap plasmid variants can be transfected into HEK293 cells, purified, and subjected to selective screening. These screens are based on the presence of vector genome DNA, rather than transgene expressions such as mRNA or protein. Capsid selection platforms typically depend on the correct genome-capsid correlation. It is essential to minimize the packaging of a genome into a mismatched capsid. Mosaic capsid formation during viral library production is also possible. It has been observed that not only the capsid itself but also the capsid-promoter interactions can contribute to CNS cell-selective gene expression in animals. Capsid protein is also involved in driving the epigenetic status of the vector genome to regulate transgene expression. Taken together, a screening platform carrying a therapeutic genome with high capsid-genome correlation is desired for SMA gene therapy. Strikingly, mosaic capsid formation and cross-packaging were limited in wild-type (WT) AAV production schemes. In other previously described capsid selection platforms (e.g., TRACER), a regulatory element from rep is used to drive cap mRNA expression during vector manufacturing. This rep element could be detrimental to capsid mRNA screening by interfering with the promoter activity. This example describes production of a WT AAV genome-like (also referred to herein as ‘WT AAV-like’) platform. Unlike other AAV library production methods that supplement rep a in trans configuration (FIG. 3A, top), in the WT AAV-like platform, rep gene was incorporated outside of the ITRs, and the promoters for cap transcription were incorporated between the ITRs of the viral genome (FIG. 3 A, bottom).

[0103] To determine vector yield, WT AAV-like plasmids were co-transfected with an adeno- helper plasmid into HEK293 cells. It was found that the vector yield is 4- to 26-fold higher than the conventional triple-plasmid transfection approach (Fig. 3B). To drive capsid gene expression Ula promoter (a ubiquitously active promoter) was used. Next, to determine if the vector packaging relied on the promoter, the Ula promoter was removed. The vector yield remained the same, indicating that the rep sequence outside of ITR drives capsid gene expression and provides the Rep proteins for vector packaging, independent of the promoter type (e.g., independent of Ula promoter). Then promoters with minimal activities were incorporated into HEK293 cells (such as the Mecp2 promoter, which is neuronal- specific, and CK8 promoter, which is muscle-specific) and the SMN1 promoter in front of the capsid gene; all these constructs surprisingly maintain high vector yield. The high vector yield may be partially due to the physical linkage of rep and cap genes on the same ITR-containing DNA molecule, which ensures the expression of Rep and Cap, and the vector genomes in the same cells. The WT AAV-like platform allows the production of both replication-defective genomes and generates capsid libraries for tissue / cell selection under promoters without rep elements.

[0104] Next to determine if the WT-AAV-like platform can reduce cross-packaging and mosaic capsid formation, the start codons of the nucleic acid sequences encoding AAV9 VP 1-3 proteins were mutated to nonsense mutations in the WT AAV9-like system to create an “AAV9-null vector” (FIG. 3C top). Plasmids encoding the AAV9-null vectors were transfected separately or at ratios of 1:1, 1:2, 1:4, 1:8, or 1:16 with adeno-helper plasmid into HEK293 cells. The ratios of the AAV9:AAV9-null vector genome were 1:0.4, 1:0.8, 1:1, 1:0.9, and 1:0.6 in the purified vectors, indicating that vector genome packaging is selective for the encoded capsid by itself, not randomly (FIG. 3C, bottom). In contrast, the vector genome composition is comparable to plasmid input when WT-AAV8-like and WT-AAV9-like plasmids were co-transfected at different ratios (FIG. 3D).

[0105] EQUIVALENTS

[0106] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0107] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0108] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0109] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0110] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0111] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0112] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

Claims

CLAIMSWhat is claimed is:

1. A nucleic acid comprising, in a 5 ' to 3 ' orientation, a Rep gene, a 5 ' inverted terminal repeat (ITR), a promoter, a Cap gene, and a 3' ITR.

2. The nucleic acid of claim 1, wherein the nucleic acid does not have an ITR that is located upstream of the Rep gene (e.g., located at the 5' end of the Rep gene).

3. The nucleic acid of claim 1 or 2, wherein the promoter is a constitutive promoter or a tissue-specific promoter.

4. The nucleic acid of any one of the preceding claims, wherein the promoter is a cTnT promoter, a chicken beta actin (CBA) promoter, a Ula promoter, a MAGI promoter, a Syn promoter, a MECP2 promoter, a CD68 promoter, a MCK7 promoter, a CK8e promoter, a TBG promoter, a Gfap promoter, or a SMN 1 promoter.

5. The nucleic acid of any one of the preceding claims wherein the 5’ ITR and the 3’ ITR are AAV2 ITRs.

6. The nucleic acid of claim 5, wherein the 5’ ITR or 3’ ITR is a mutant ITR.

7. The nucleic acid of any one of the preceding claims, wherein the Rep gene encodes one or more AAV viral replication proteins, optionally one or more of Rep78, Rep68, Rep52, and Rep40.

8. The nucleic acid of any one of the preceding claims, wherein the Cap gene encodes an AAV capsid protein.

9. A method of producing a capsid protein, the method comprising transfecting a host cell with the nucleic acid (e.g., WT AAV-like vector) of any one of the preceding claims.

10. A method of producing a capsid protein, the method comprising administering the nucleic acid of any one of claims 1 to 8 to a subject.

11. The method of claim 10, wherein the subject is a mammalian subject.

12. The method of claim 10 or 11, wherein the subject is a human, a mouse, a rat, a monkey, or a pig.

13. An AAV capsid library comprising a plurality of unique WT AAV-like vectors; wherein each WT AAV-like vector comprises, in a 5' to 3' orientation, a Rep gene, a 5' inverted terminal repeat (ITR), a promoter, a Cap gene, and a 3' ITR; and wherein the Cap gene of each WT AAV-like vector encodes a unique AAV capsid protein, relative to the other WT AAV-like vectors of the plurality.

14. The AAV capsid library of claim 13 comprising 10-1000 unique WT AAV-like vectors, each unique WT AAV-like vector comprising a unique AAV capsid protein.

15. The AAV capsid library of claim 13 or 14, wherein the unique AAV capsid protein of each WT AAV-like vector has at least one amino acid insertion, substitution, or deletion relative to at least one other unique AAV capsid protein of the library.

16. The AAV capsid library of any one of claims 13 to 15, wherein the unique WT AAV-like vectors do not have an ITR that is located upstream of the Rep gene (e.g., located at the 5' end of the Rep gene).

17. The AAV capsid library of any one of claims 13 to 16, wherein the promoter is a constitutive promoter or a tissue- specific promoter.

18. The AAV capsid library of any one of claims 13 to 17, wherein the promoter is a cTnT promoter, a chicken beta actin (CBA) promoter, a Ula promoter, a MAGI promoter, a Syn promoter, a MECP2 promoter, a CD68 promoter, a MCK7 promoter, a CK8e promoter, a TBG promoter, a Gfap promoter, or a SMN 1 promoter.

19. The AAV capsid library of any one of claims 13-18, wherein the Rep gene encodes one or more AAV viral replication proteins, optionally one or more of Rep78, Rep68, Rep52 and Rep40.

20. A method of screening WT AAV-like vectors in vivo, the method comprising administering the AAV capsid library of any one of claims 13 to 19 to one or more subjects.

21. The method of claim 20, wherein the one or more subjects are one or more mammalian subjects.

22. The method of claim 20 or 21, wherein the one or more subjects are one or more humans, mice, rats, monkeys, or pigs.

23. The method of any one of claims 20 to 22, wherein the unique AAV capsid protein of each of the unique WT AAV-like vectors is preferentially expressed in a target tissue.

24. The method of any one of claims 20 to 23 further comprising isolating the unique capsid proteins from the subject.

25. A recombinant adeno-associated virus (rAAV) particle comprising:(i) a recombinant adeno-associated virus (rAAV) vector; and(ii) a capsid protein isolated from a subject according to claim 24.

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