Tissue-targeted modified AAV capsids and methods of use thereof

Abstract

Provided herein is a modified AAV capsid comprising modified AAV capsid protein with improved transduction in muscle tissue, reduced immunogenicity, reduced neutralizing antibody binding, reduced liver tissue transduction, and combinations thereof.Also provided is a method of treating a patient with an AAV viral vector comprising modified AAV capsid protein.In some embodiments, the capsid protein is VP1, VP2 or VP3 capsid protein.In some embodiments, modified VR VIII comprises peptide insertion.

Description

[Technical field]

[0001] Field of Disclosure The present disclosure is directed to molecular biology, gene therapy, and compositions and methods for enhancing transduction of AAV capsids comprising modified AAV capsid proteins. In particular, modified AAV capsids comprising modified AAV capsid proteins are provided that have enhanced transduction in is muscle tissue and / or reduced transduction in liver tissue.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 178,965, filed April 23, 2021, and U.S. Provisional Patent Application No. 63 / 299,697, filed January 14, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0003] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the text file named "LOCN_011_001WO_SeqList_ST25", created on April 21, 2022, and sized at 1.33 MB, are hereby incorporated by reference in their entirety into this specification. [Background technology]

[0004] background Adeno-associated virus (AAV) vector-based therapeutics can have limited transduction in certain tissue types, and transduction can be limited due to pre-existing neutralizing antibodies (NAbs) specific for certain AAV serotypes.

[0005] In particular, AAV8 and AAV9 are commonly used vectors for therapies delivered by systemic injection for skeletal muscle transduction.However, these serotypes also transduce the liver, and a significant percentage of the patient population has moderate to high NAb titers against AAV8 or AAV9.Therefore, there is a need to develop AAV capsids with modified AAV capsid proteins that have enhanced tissue-specific transduction in desired tissues and / or reduced transduction in undesired tissues.In some embodiments, it is desirable to have enhanced transduction in muscle tissues and / or reduced transduction in liver tissues.

[0006] Disclosed herein is an AAV capsid that is composed of modified and chimeric AAV capsid protein sequences that have enhanced muscle, transduction, and reduced transduction in liver tissue.In some aspects, the AAV capsid is composed of modified and chimeric AAV capsid protein sequences that reduce sensitivity to pre-existing neutralizing antibodies. Summary of the Invention [Means for solving the problem]

[0007] overview The present disclosure provides a modified AAV capsid protein comprising a modified variable region (VR) VIII. In some embodiments, the capsid protein is a VP1, VP2 or VP3 capsid protein. In some embodiments, the modified VR VIII comprises a peptide insertion.

[0008] In some embodiments, the peptide insertion comprises an RGD motif peptide insertion. In some embodiments, the RGD motif insertion comprises RGDLGLS (SEQ ID NO: 303), RGDLSTP (SEQ ID NO: 304), SNSRGDYNSL (SEQ ID NO: 305), ENRRGDFNNT (SEQ ID NO: 306), SRGDYNSL (SEQ ID NO: 307), RGDYNSL (SEQ ID NO: 308), RGDLST (SEQ ID NO: 309) or RGDYVGL (SEQ ID NO: 310).

[0009] In some embodiments, the RGD motif insertion comprises a camelid heavy chain only antibody variable domain (VHH) RGD peptide. In some embodiments, the VHH RGD peptide has the amino acid sequence [ka] and comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to

[0010] In some embodiments, the peptide insertion comprises an acetylcholinesterase collagenoplastic tail (ColQ) peptide. In some embodiments, the ColQ peptide has the amino acid sequence [ka] and comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to

[0011] In some embodiments, the peptide insertion comprises one or more linker sequences at the N-terminus or C-terminus of the inserted peptide. In some embodiments, the linker sequence comprises GGGGS (SEQ ID NO:311), GGGGSGGGGS (SEQ ID NO:312), GGGGSGGGGSGGGGGS (SEQ ID NO:313), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:314), or GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:315).

[0012] In some embodiments, the AAV serotype is AAV9 or AAVRh74.

[0013] In some embodiments, the modified AAV capsid protein further comprises one or more amino acid mutations. In some embodiments, the one or more amino acid mutations reduce liver transduction. In some embodiments, the mutations comprise i) at least one of F503I, G507I, Y707C, and / or Y708C of Rh74, or ii) N498I of AAV9.

[0014] In some embodiments, the capsid protein comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53, 75, 102, 124, 245, 249, 255, 258, 262, or 267.

[0015] The present disclosure provides modified AAV capsid proteins comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53, 75, 102, 124, 245, 249, 255, 258, 262, or 267.

[0016] In some aspects, the capsid protein is encoded by a nucleic acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 151, 173, 199, 221, 270, 274, 280, 283, 287, or 292.

[0017] The present disclosure provides an AAV capsid comprising one or more AAV capsid proteins according to any embodiment of the present disclosure.

[0018] In some embodiments, AAV capsid has enhanced transduction in targeted tissue or cell type compared to other tissue or cell type.In some embodiments, targeted tissue type is muscle tissue or muscle cell.In some embodiments, AAV capsid has enhanced transduction in muscle tissue.In some embodiments, AAV capsid has reduced transduction in non-targeted tissue or cell type.In some embodiments, non-targeted tissue includes liver, lung, kidney, brain, spleen, intestine, spinal cord, or reproductive organs.

[0019] In some embodiments, transduction in muscle tissue is enhanced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 100%, about 200%, or about 300% compared to the parental and / or unmodified AAV capsid.

[0020] In some embodiments, the capsid comprises i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:53, and ii) a VP2 / VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:102.

[0021] In some embodiments, the capsid comprises i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:75, and ii) a VP2 / VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:124.

[0022] In some embodiments, the capsid comprises i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:245, and ii) a VP2 / VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:258.

[0023] In some embodiments, the capsid comprises i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:249, and ii) a VP2 / VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:262.

[0024] In some embodiments, the capsid comprises i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:255, and ii) a VP2 / VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:267.

[0025] The present disclosure provides vectors comprising a nucleic acid sequence encoding the modified AAV capsid proteins of the present disclosure.

[0026] In some aspects, the nucleic acid sequence encoding the modified AAV capsid protein is operably linked to regulatory elements that control expression of the capsid protein in a host cell.

[0027] The present disclosure provides an AAV viral vector comprising a modified capsid protein of this disclosure or an AAV capsid of this disclosure.

[0028] In some aspects, the AAV viral vector comprises a recombinant AAV (rAAV) vector encoding a therapeutic transgene or nucleotide sequence of interest (NOI).

[0029] The present disclosure provides a cell comprising a vector of the present disclosure or an AAV viral vector of the present disclosure.

[0030] The present disclosure provides a pharmaceutical composition comprising an AAV viral vector of the present disclosure and at least one pharma- ceutically acceptable excipient and / or additive.

[0031] The present disclosure provides a method of providing a therapeutic transgene or protein to a subject, the method comprising administering to the subject an AAV viral vector of the present disclosure or a pharmaceutical composition of the present disclosure.

[0032] The present disclosure provides a method for treating a subject with disease and / or disorder, comprising administering to the subject at least one therapeutically effective amount of the AAV viral vector of the present disclosure or the pharmaceutical composition of the present disclosure.In some embodiments, the disease and / or disorder is a muscle and / or neuromuscular disorder.In some embodiments, the muscle and / or neuromuscular disorder is a muscular dystrophy or myotonic dystrophy.

[0033] In some aspects, the AAV viral vector or pharmaceutical composition is administered to the subject intravenously, intrathecally, intracerebrally, intravenously, intranasally, intratracheally, intraaurally, intraocular or periocular, orally, rectally, transmucosally, by inhalation, transdermal, parenterally, subcutaneously, intradermally, intramuscularly, intracisternally, intraneurally, intrapleurally, topically, intralymphatically, intracisternally, or intraneurally. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 is a schematic diagram listing modified muscle-targeted AAV7 and AAVRh74 chimeric capsid proteins.

[0035] [Diagram 2] FIG. 2 is a schematic depicting a muscle-targeted AAV Rh8 variant capsid containing liver-detargeting amino acid mutations.

[0036] [Diagram 3] FIG. 3 is a schematic depicting the modified muscle-targeted AAV capsid protein of the present disclosure.

[0037] [Figure 4] FIG. 4 is a schematic depicting the modified muscle-targeted AAVRh74 capsid protein of the present disclosure.

[0038] [Diagram 5] FIG. 5 shows immunofluorescence images demonstrating the transduction efficiency of HEK293 cells with AAV vectors containing a targeting peptide that specifically displays VP1.

[0039] [Figure 6-1] 6A-6D show immunofluorescence images demonstrating improved transduction efficiency of C2C12 cells with modified muscle-targeted AAV capsids. [Figure 6-2] Same as above.

[0040] [Figure 7-1] Figures 7A-C show the results of ex vivo imaging of AAV9, LBV30, and LBV31. Figure 7A is an ex vivo immunofluorescence image showing in vivo targeting of AAV9, LBV30, and LBV31 capsids in mice. Figures 7B and 7C are graphs quantifying immunofluorescence in various tissues. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0041] [Figure 8] FIG. 8 is a series of schematic diagrams depicting RGD peptide insertion scaffolds and approaches.

[0042] [Figure 9] 9 is a graph depicting transduction of AAV viral vectors of the present disclosure in the indicated mouse tissues: liver, heart, lung, spleen, kidney, intestine, testis, tongue, quadriceps (quad), gastrocnemius (gc), tibialis anterior (ta), diaphragm, and brain. The y-axis represents luminescence of a capsid-encapsulated luciferase reporter vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description The present disclosure provides gene therapy compositions comprising modified and chimeric AAV capsid proteins for delivery of packaged therapeutic agents to muscle tissue.

[0044] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses related to this name, belonging to the Parvoviridae family, Dependoparvovirus genus. Adeno-associated virus is a single-stranded DNA virus that grows in cells where certain functions are provided by the co-infection of a helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169- 228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews can be applied to additional AAV serotypes characterized after the publication date of these reviews, because it is well known that the various serotypes are very closely related, both structurally and functionally, and even at the genetic level. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974).) For example, all AAV serotypes appear to exhibit very similar replication properties mediated by homologous rep genes, and all have three related capsid proteins, such as those expressed in AAV2. This similarity is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends that correspond to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes can infect cells from a variety of tissue types. At least eleven AAV serotypes, which are numbered consecutively, are known in the art.Non-limiting exemplary serotypes useful in the methods disclosed herein include any of eleven serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV PHP.B. AAV particles comprise, consist essentially of, or consist of three major viral proteins: VP1, VP2, and VP3. In some embodiments, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPO1, AAVPHP.B, AAVrh74, or AAVrh.10.

[0045] Exemplary adeno-associated and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPO1, AAVPHP.B, AAVrh74, and AAVrh.10). Exemplary adeno-associated and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV), and AAV hybrids that contain the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (described in detail in Kerun et al. JCI Insight, 2019; 4(22):e131610), and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018; 26(1): 289-303). AAV Structure and Function

[0046] AAV is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length and contains two 145 nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided under GenBank Accession No. NC_l829; the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829; the AAV-5 genome is provided under GenBank Accession No. AF085716; the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively; and the AAV-9 genome is provided under Gao et al., J. Virol., 78: 6381-6388 (2004); AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928. U.S. Pat. No. 9,434,928 also provides the sequences of capsid proteins and self-complementary genomes. In one embodiment, the AAV genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITR. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19) coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the generation of four rep proteins from the rep gene (rep 78, rep 68, rep 52, and rep 40). The rep proteins have multiple enzymatic properties that are ultimately responsible for the replication of the viral genome.

[0047] The cap gene is expressed from the p40 promoter and encodes three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of the three related capsid proteins. More specifically, after a single mRNA is transcribed into which each of the VP1, VP2, and VP3 proteins is translated, it can be spliced ​​in two different ways: either a longer intron or a shorter intron can be excised, resulting in the formation of two pools of mRNAs: a 2.3 kb-long and a 2.6 kb-long mRNA pool. The longer intron is often preferred, and therefore the 2.3 kb-long mRNA can be referred to as the major splice variant. This form lacks the first AUG codon at which synthesis of the VP1 protein begins, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon remaining in the major splice variant is the start codon for the VP3 protein. However, upstream of that codon in the same open reading frame is an ACG sequence (encoding a threonine) that is surrounded by an optimal Kozak (translation start) context.Becerra SP et al., (December 1985). "Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon". Proceedings of the National Academy of Sciences of the United States of America. 82 (23): 7919-23, Cassinotti P et al., (November 1988). "Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced ​​mRNA coding for virus capsid protein 1". Virology. 167 (1): 176-84, Muralidhar S et al., (January 1994). "Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity". Journal of Virology. 68 (1): 170-6, and Trempe JP, Carter BJ (September 1988). "Alternate mRNA splicing is required for synthesis of adeno-associated viral VP1 capsid protein". Journal of Virology. 62 (9): 3356-63, each of which is incorporated herein by reference, contributes to low-level synthesis of the VP2 protein, which, like VP1, is actually a VP3 protein with additional N-terminal residues. A single consensus polyA site is located at map position 95 of the AAV genome.The life cycle and genetic characteristics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0048] Each VP1 protein contains a VP1 portion, a VP2 portion, and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein that is unique to the VP1 protein. The VP2 portion is an amino acid sequence present in the VP1 protein that is also found in the N-terminal portion of the VP2 protein. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is the C-terminal portion of the VP1 protein that is shared with the VP1 and VP2 proteins.

[0049] The VP3 protein can be further divided into separate variable surface regions I to IX (VRI to IX, also referred to as VR1 to VR8). Each of the variable surface regions (VRs) can include or contain a specific amino acid sequence that can confer a unique infection phenotype to a particular serotype (e.g., reduced antigenicity, improved transduction and / or tissue-specific tropism compared to other AAV serotypes), either alone or in combination with the specific amino acid sequences of each of the other VRs, as described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9" J. Virol., Vol. 86 (12): 6947-6958, June 2012, the contents of which are incorporated herein by reference.

[0050] AAV has unique features that make it attractive as a vector to deliver foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is asymptomatic and asymptomatic. Moreover, AAV infects many mammalian cells, thereby allowing the possibility of targeting many different tissues in vivo. Moreover, AAV can slowly transduce dividing and non-dividing cells and persist as transcriptionally active nuclear episomes (extrachromosomal elements) essentially for the life of these cells. The AAV proviral genome is inserted as cloned DNA into plasmids, thus making the construction of recombinant genomes feasible. Moreover, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb genome (encoding the replication and structural capsid proteins, rep-cap,) can be replaced with foreign DNA to generate AAV vectors. The rep and cap proteins can be provided in trans. Another significant feature of AAV is that it is an extremely stable and hardy virus. AAV easily survives the conditions used to inactivate adenovirus (56°C-65°C for several hours), making cryopreservation of AAV less important. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to superinfection.

[0051] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. Clark et al., Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 8098-8108 (1996). See also, Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction results in the appearance of transgene products in the systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94: 13921- 13926 (1997).Furthermore, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors required for proper antibody glycosylation, folding and secretion, indicating that muscle can stably express secreted protein therapeutics.The recombinant AAV (rAAV) genome of the present invention comprises, consists essentially of, or consists of a nucleic acid molecule encoding a therapeutic protein and one or more AAV ITRs flanking the nucleic acid molecule. The production of pseudotyped rAAV is disclosed, for example, in WO2001083692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated.See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art. Recombinant AAV vectors

[0052] "rAAV vector" as used herein refers to a vector that comprises, consists essentially of, or consists of one or more transgene sequences and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors, when present in a host cell that provides the functionality of the rep and cap gene products, for example by transfection into the host cell, can be replicated and packaged into infectious viral particles that contain the modified AAV capsid proteins of the present disclosure. In some embodiments, the AAV vector contains a promoter, at least one nucleic acid that can code for at least one protein or RNA, and / or enhancer and / or terminator within the flanking ITRs that are packaged into infectious AAV particles. The nucleic acid portion that is packaged into the capsid can be referred to as the AAV vector genome. The plasmid that contains the rAAV vector can also contain elements for production, such as antibiotic resistance genes, replication origin sequences, etc., but these are not packaged into the capsid and therefore do not form part of the AAV particle.

[0053] In some embodiments, the rAAV vector may comprise at least one transgene nucleic acid molecule. In some embodiments, the rAAV vector may comprise at least one AAV inverted terminal repeat (ITR) sequence. In some embodiments, the rAAV vector may comprise at least one promoter sequence. In some embodiments, the rAAV vector may comprise at least one enhancer sequence. In some embodiments, the rAAV vector may comprise at least one polyA sequence. In some embodiments, the rAAV vector may comprise at least one reporter protein.

[0054] In some embodiments, the rAAV vector can include a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the rAAV vector can include, from the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence.

[0055] In some embodiments, rAAV vector can contain more than one transgene nucleic acid molecule.In some embodiments, rAAV vector can contain at least two transgene nucleic acid molecules, thus rAAV vector contains a first transgene nucleic acid molecule and at least a second transgene nucleic acid molecule.In some embodiments, the first and at least a second transgene nucleic acid molecule can contain the same nucleic acid sequence.In some embodiments, the first and at least a second transgene nucleic acid molecule can contain different nucleic acid sequences.In some embodiments, the first and at least a second transgene nucleic acid sequence can be adjacent to each other.

[0056] In some embodiments, the rAAV vector may include more than one promoter sequence. In some embodiments, the rAAV vector may include at least two promoter sequences, thus the rAAV vector includes a first promoter sequence and at least a second promoter sequence. In some embodiments, the first and at least a second promoter sequence may include the same sequence. In some embodiments, the first and at least a second promoter sequence may include different sequences. In some embodiments, the first and at least a second promoter sequence may be adjacent to each other. In some embodiments, where the rAAV vector also includes a first transgene nucleic acid molecule and at least a second transgene nucleic acid molecule, the first promoter may be located upstream (5') of the first transgene nucleic acid molecule, and the at least a second promoter may be located between the first transgene nucleic acid molecule and the at least a second transgene nucleic acid molecule, thus the at least a second promoter is downstream (3') of the first transgene nucleic acid molecule and upstream (5') of the at least a second transgene nucleic acid molecule.

[0057] Any of the rAAV vectors may further comprise at least one enhancer. The at least one enhancer may be located anywhere within the rAAV vector. In some embodiments, the at least one enhancer may be located immediately upstream (5') of the promoter. Thus, the rAAV vector may comprise, in the 5' to 3' direction, a first AAV ITR sequence, an enhancer, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the at least one enhancer may be located immediately downstream (3') of the promoter. Thus, the rAAV vector may comprise, in the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, an enhancer, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the at least one enhancer may be located immediately downstream of the transgene nucleic acid molecule. Thus, a rAAV vector can include, from the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, an enhancer, a polyA sequence, and a second AAV ITR sequence.

[0058] The rAAV vector of the present disclosure may include any transgene nucleic acid molecule known in the art. In some aspects, the transgene nucleic acid is a therapeutic transgene. In some aspects, the transgene nucleic acid molecule is interchangeably referred to as a nucleotide sequence of interest (NOI). The NOI includes, without limitation, any nucleotide sequence or transgene that can be delivered by a vector. The NOI may be synthetic, may be derived from naturally occurring DNA or RNA, may be codon optimized, may be recombinant RNA / DNA, may be cDNA, may be partial genomic DNA, and / or may be a combination thereof. The NOI may be, but need not be, a coding region or partial coding region. The NOI may be RNA / DNA in a sense or antisense orientation. The NOI is also referred to herein, without limitation, as a transgene, a heterologous sequence, a gene, a therapeutic gene. The NOI may also encode a POI (protein of interest), a partial POI, a mutated version or variant of a POI. The POI may be similar to or correspond to a wild-type protein. The POI may be a fusion protein or a nucleoprotein complex, such as a CRISPR / Cas nucleoprotein complex. The POI may be a PUF or PUMBY protein. In some embodiments, the POI may be an RNA targeting or RNA binding protein or a nucleoprotein complex. The NOI or transgene, including the unguided RNA binding fusion protein.

[0059] In some embodiments, the NOI is a nucleic acid encoding a target RNA-binding fusion protein that is not a target RNA-guided fusion protein and therefore contains at least one RNA-binding polypeptide that can bind to a target RNA without a corresponding gRNA sequence. Such non-guided RNA-binding polypeptides include, without limitation, at least one RNA-binding protein or an RNA-binding portion thereof that is a PUF (Pumilio and FBF homology family) protein. This type of RNA-binding polypeptide can be used instead of a gRNA-guided RNA-binding protein such as CRISPR / Cas. The unique RNA recognition mode of PUF proteins (derived from Drosophila Pumilio and C.elegans fem-3 binding factor), which are involved in mediating mRNA stability and translation, is well known in the art. The PUF domain of human Pumilio1, also known in the art, binds tightly to cognate RNA sequences and its specificity can be modified. The PUF domain of human Pumilio1 contains eight PUF modules that recognize eight consecutive RNA bases, each module recognizing a single base. Two amino acid side chains in each module recognize the Watson-Crick ends of corresponding bases, determining the specificity of that module, so that PUF modules can be designed to specifically bind RNAs up to 8-16 nt in length. Wang et al., Nat Methods. 2009; 6 (11): 825-830. See also WO2012 / 068627, which is incorporated herein by reference in its entirety.

[0060] The modular nature of PUF-RNA interactions has been used to rationally engineer the binding specificity of PUF domains (Cheong, CG & Hall, TM (2006) PNAS 103: 13635-13639; Wang, X. et al (2002) Cell 110: 501-512). However, only successful design of PUF proteins with modules that recognize adenine, guanine or uracil has been reported prior to the teachings of WO2012 / 06827, supra. Wild-type PumHD does not bind cytosine (C), but it has been shown that by molecular engineering, some of the Pum units can be mutated to bind C with good yield and specificity. See, e.g., Dong, S. et al. Specific and modular binding code for cytosine recognition in Pumilio / FBF (PUF) RNA-binding domains, The Journal of biological chemistry 286, 26732-26742 (2011). Thus, PumHD is a modified version of the WT Pumilio protein that exhibits programmable binding to any 8-base sequence of RNA. Each of the 8 units of PumHD can bind to all four RNA bases, and RNA bases flanking the target sequence do not affect binding.See also the following references for art-recognized RNA binding rules for PUF design:Filipovska A, Razif MF, Nygard KK, & Rackham O. A universal code for RNA recognition by PUF proteins. Nature chemical biology, 7(7), 425-427 (2011);Filipovska A, & Rackham O. Modular recognition of nucleic acids by PUF, TALE and PPR proteins. Molecular BioSystems, 8(3), 699-708 (2012);Abil Z, Denard CA, & Zhao H. Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA. Journal of biological engineering, 8(1), 7 (2014);Zhao Y, Mao M, Zhang W, Wang J, Li H, Yang Y, Wang Z, & Wu J. Expanding RNA binding specificity and affinity of engineered PUF domains. Nucleic Acids Research, 46(9), 4771-4782 (2018);Shinoda K, Tsuji S, Futaki S, & Imanishi M. Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation. ChemBioChem, 19(2), 171-176 (2018);Koh YY, Wang Y, Qiu C, Opperman L, Gross L, Tanaka Hall TM, & Wickens M. Stacking Interactions in PUF-RNA Complexes. RNA, 17(4), 718-727 (2011).

[0061] Thus, it is well known in the art that human PUM1 (1186 amino acids) contains an RNA-binding domain (RBD) (also known as Pumilio homology domain PUM-HD amino acids 828 to amino acids 1175) at the C-terminus of the protein, and that PUFs are based on the RBD of human PUM1. There are eight structural repeat modules of 36 amino acids for RNA binding (except module 7, which has 43 amino acids), as well as flanking N- or C-terminal regions important for protein structure and stability. Within each repeat module, amino acids 12, 13, and 16 are important for RNA binding, with 12 and 16 involved in RNA base recognition. Amino acid 13 can be modified to stack with the RNA base and tune specificity and affinity. Alternatively, the PUF design can maintain amino acid 13 as the native residue of human PUM1. In some embodiments of the PUF or PUMBY compositions disclosed herein, amino acid 13 will be engineered with H (for stacking), and in other embodiments, with Y. In some embodiments, stacking residues may be modified to improve binding and specificity. Recognition is performed in the opposite orientation from N-terminus to C-terminus PUFs recognizing RNA from 3' to 5'. Thus, 8-module (8PUF) PUF engineering known in the art mimics human proteins. An exemplary 8-mer RNA recognition (8PUF) would be designed as follows: R1'-R1-R2-R3-R4-R5-R6-R7-R8-R8'. In one embodiment, 8PUF is used as RBD. In another embodiment, variations of the 8PUF design are used to create 14-mer RNA recognition (14PUF) RBD, 15-mer RNA recognition (15PUF) RBD, or 16-mer RNA recognition (16PUF) RBD. In another embodiment, the PUF can be engineered to contain a 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 24-mer, 30-mer, 36-mer, or any number of modules in between.Shinoda et al., 2018; Criscuolo et al., 2020. See also U.S. Patent 9,580,714, which is incorporated herein in its entirety.

[0062] In some embodiments of the unguided RNA-binding fusion protein of the present disclosure, the fusion protein comprises at least one RNA-binding protein or an RNA-binding portion thereof, which is a PUMBY (Pumilio-based assembly) protein. The RNA-binding protein PumHD, which is widely used in native and modified forms to target RNA, has been engineered into a designed protein structure that results in a set of four standard protein modules, each targeting one RNA base. These modules (i.e., Pumby for Pumilio-based assembly) are linked to strands of various compositions and lengths to bind to the desired target RNA. In essence, PUMBY is a simpler, modular form of PumHD, where single protein units of PumHD are linked into an array of any size and binding sequence specificity. The specificity of such Pumby-RNA interactions is high, with undetectable binding of Pumby strands to RNA sequences with three or more mismatches from the target sequence. Katarzyna et al., PNAS, 2016; 113 (19): E2579-E2588. See also US2016 / 0238593, which is incorporated by reference in its entirety.

[0063] In some embodiments of the composition of the present disclosure, the first RNA binding protein comprises a Pumilio and FBF (PUF) protein. In some embodiments, the first RNA binding protein comprises a Pumilio-based assembly (PUMBY) protein. In some embodiments, the PUF or PUMBY RNA binding protein is fused with a nuclease domain, such as the zinc finger endonuclease known as ZC3H12A (E17).

[0064] In some embodiments of the composition of the present disclosure, at least one of the RNA-binding proteins or its RNA-binding portion is a PPR protein. PPR proteins (proteins with pentatricopeptide repeat (PPR) motifs from plants) are nuclear-encoded and exclusively control genes that act specifically on organelles (chloroplasts and mitochondria), cutting, translation, splicing, RNA editing, and RNA stability at the RNA level. PPR proteins generally have a structure of 35 amino acid motifs, with the PPR motif being about 10 consecutive amino acids. Combinations of PPR motifs can be used for sequence-selective binding to RNA. PPR proteins are often composed of about 10 repeat domains of PPR motifs. The PPR domain or RNA-binding domain can be configured to be catalytically inactive. WO2013 / 058404, which is incorporated herein by reference in its entirety.

[0065] In some embodiments, the fusion protein disclosed herein comprises a linker between at least two RNA-binding polypeptides. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises one or more repeats of the tripeptide GGS. In other embodiments, the linker is a non-peptide linker. In some embodiments, the non-peptide linker comprises polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene / propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharide, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylate, lipid polymer, chitin, hyaluronic acid, heparin, or an alkyl linker.

[0066] In some embodiments, at least one RNA binding protein does not require multimerization for RNA binding activity. In some embodiments, at least one RNA binding protein is not a monomer of a multimeric complex. In some embodiments, the multimeric protein complex does not include an RNA binding protein. In some embodiments, at least one RNA binding protein selectively binds to a target sequence in an RNA molecule. In some embodiments, at least one RNA binding protein does not include affinity for a second sequence in an RNA molecule. In some embodiments, at least one RNA binding protein does not include high affinity or selectively bind to a second sequence in an RNA molecule. In some embodiments, at least one RNA binding protein includes between 2 amino acids and 1300 amino acids, including the end.

[0067] In some embodiments, at least one RNA binding protein of the fusion protein disclosed herein further comprises a sequence encoding a nuclear localization signal (NLS). In some embodiments, the nuclear localization signal (NLS) is located at the N-terminus of the RNA binding protein. In some embodiments, at least one RNA binding protein comprises an NLS at the C-terminus of the protein. In some embodiments, at least one RNA binding protein further comprises a first sequence encoding a first NLS and a second sequence encoding a second NLS. In some embodiments, the first NLS or the second NLS is located at the N-terminus of the RNA binding protein. In some embodiments, at least one RNA binding protein comprises a first NLS or a second NLS at the C-terminus of the protein. In some embodiments, at least one RNA binding protein further comprises a NES (nuclear export signal) or other peptide tag or secretion signal. In some embodiments, the tag is a FLAG tag.

[0068] In some embodiments, the fusion proteins disclosed herein comprise at least one RNA binding protein as a first RNA binding protein together with a second RNA binding protein that comprises or consists of a nuclease domain.

[0069] In some embodiments, the second RNA-binding polypeptide is configured to be operable to the first RNA-binding polypeptide at the C-terminus of the first RNA-binding polypeptide. In some embodiments, the second RNA-binding polypeptide is configured to be operable to the first RNA-binding polypeptide at the N-terminus of the first RNA-binding polypeptide. In one embodiment, an exemplary fusion protein is a first RNA-binding protein based on PUF or PUMBY fused to a second RNA-binding protein that is a zinc finger endonuclease known as ZC3H12A. NOI or transgene comprising a guide RNA for an RNA-guided RNA-binding protein

[0070] In a Cas-based RNA-targeted gene therapy system, the NOI or transgene comprises a guide RNA.

[0071] The terms guide RNA (gRNA) and single guide RNA (sgRNA) are used interchangeably throughout this disclosure.

[0072] A guide RNA (gRNA) of the present disclosure may be composed of a spacer sequence and a "direct repeat" (DR) sequence. In some embodiments, the guide RNA is a single guide RNA (sgRNA) that includes a spacer sequence and a DR sequence contiguous. In some embodiments, the spacer sequence and the DR sequence are not contiguous. In some embodiments, the gRNA includes a DR sequence. The DR sequence refers to a repeated sequence in a CRISPR locus (naturally occurring in a bacterial genome or a plasmid) that is interspersed with a spacer sequence. It is well known that if the sequence of the relevant CRISPR locus is known, the DR sequence of the corresponding (or cognate) Cas protein can be inferred. In some embodiments, the guide RNA includes a direct repeat (DR) sequence and a spacer sequence. In some embodiments, the sequence encoding the guide RNA or single guide RNA of the present disclosure includes or consists of a spacer sequence and a DR sequence separated by a linker sequence. In some embodiments, the linker sequence may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number of nucleotides (nt) in between. In some embodiments, the linker sequence may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number of nucleotides in between. In some embodiments, the DR sequence is a Cas13d DR sequence.

[0073] In one embodiment, the gRNA that hybridizes with one or more target RNA molecules in a Cas13d-mediated manner comprises one or more direct repeat (DR) sequences, one or more spacer sequences, such as one or more sequences comprising an array of DR-spacer-DR-spacer. In one embodiment, multiple gRNAs are generated from a single array, where each gRNA can be different, e.g., target different RNAs, or multiple target regions from a single RNA, or combinations thereof. In some embodiments, the isolated gRNA comprises one or more direct repeat sequences, e.g., unprocessed (e.g., about 36 nt) or processed DR (e.g., about 30 nt). In some embodiments, the gRNA can further comprise one or more spacer sequences specific to the target RNA (e.g., complementary to the target RNA). In certain such embodiments, multiple polIII promoters can be used to drive multiple gRNAs, spacers and / or DRs. In one embodiment, the guide array comprises DR(about 36 nt)-spacer(about 30 nt)-DR(about 36 nt)-spacer(about 30 nt).

[0074] The guide RNA (gRNA) of the present disclosure may include non-naturally occurring nucleotides. In some embodiments, the guide RNA of the present disclosure, or the sequence encoding the guide RNA, comprises or consists of modified or synthetic RNA nucleotides. Exemplary modified RNA nucleotides include, but are not limited to, pseudouridine (Ψ), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G), hypoxanthine, xanthine, xanthosine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-methylcytidine, 5-hydroxymethylcytosine, isoguanine, and isocytosine.

[0075] The guide RNA (gRNA) of the present disclosure can bind to modified RNA in target sequence. The guide RNA (gRNA) of the present disclosure can bind to modified or mutant (e.g., pathogenic) RNA in target sequence. Exemplary epigenetically or post-transcriptionally modified RNA includes, but is not limited to, 2'-O-methylated (2'-OMe) (2'-O-methylated is present on the oxygen of the free 2'-OH of ribose moiety), N6-methyladenosine (m6A), and 5-methylcytosine (m5C).

[0076] In some embodiments of the composition of the present disclosure, the guide RNA of the present disclosure comprises at least one sequence encoding a non-coding C / D box small nucleolar RNA (snoRNA) sequence. In some embodiments, the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, where the target sequence of the RNA molecule comprises at least one 2'-OMe. In some embodiments, the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, where the at least one sequence that is complementary to the target RNA comprises a box C motif (RUGAUGA) and a box D motif (CUGA).

[0077] The spacer sequences of the present disclosure bind to a target sequence of an RNA molecule. In some embodiments, the spacer sequences of the present disclosure bind to a pathogenic target RNA.

[0078] In some embodiments of the compositions of the present disclosure, the sequence comprising the gRNA further comprises a spacer sequence that specifically binds to the target RNA sequence. In some embodiments, the spacer sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99%, or any percentage therebetween complementary to the target RNA sequence. In some embodiments, the spacer sequence has 100% complementary to the target RNA sequence. In some embodiments, the spacer sequence comprises or consists of 20 nucleotides. In some embodiments, the spacer sequence comprises or consists of 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, or 29 nucleotides. In some embodiments, the spacer sequence comprises or consists of 26 nucleotides. In some embodiments, the spacer sequence is unprocessed and comprises or consists of 30 nucleotides. In some embodiments, the unprocessed spacer sequence comprises or consists of 30-36 nucleotides.

[0079] The DR sequence of the present disclosure binds to the Cas polypeptide of the present disclosure. When the spacer sequence of the gRNA binds to the target RNA sequence, the Cas protein bound to the DR sequence of the gRNA is positioned to the target RNA sequence. A DR sequence with sufficient complementarity to its cognate Cas protein or nucleic acid will selectively bind to the target nucleic acid sequence of the Cas protein and have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96, 97%, 98%, 99%, or any percentage therebetween identity to the sequence. In some embodiments, a sequence with sufficient complementarity has 100% identity. In some embodiments, the DR sequence of the present disclosure comprises a secondary or tertiary structure. Exemplary secondary structures include, but are not limited to, helices, stem loops, bulges, tetraloops, and pseudoknots. Exemplary tertiary structures include, but are not limited to, A-type helix, B-type helix, and Z-type helix. Exemplary tertiary structures include, but are not limited to, twisted or spiral stem loops. Exemplary tertiary structures include, but are not limited to, twisted or spiral pseudoknots. In some embodiments, the DR sequence of the present disclosure comprises at least one secondary structure or at least one tertiary structure. In some embodiments, the DR sequence of the present disclosure comprises one or more secondary structures or one or more tertiary structures.

[0080] In some embodiments of the composition of the present disclosure, guide RNA or its part selectively binds to the tetraloop motif in the RNA molecule of the present disclosure.In some embodiments, the target sequence of the RNA molecule comprises a tetraloop motif.In some embodiments, the tetraloop motif is a "GRNA" motif that comprises or consists of one or more of the following sequences: GAAA, GUGA, GCAA or GAGA.

[0081] In some embodiments of the composition of the present disclosure, the guide RNA or the portion thereof that binds to the target sequence of the RNA molecule hybridizes with the target sequence of the RNA molecule.In some embodiments, the guide RNA or the portion thereof that binds to the first RNA binding protein or the second RNA binding protein is covalently bound to the first RNA binding protein or the second RNA binding protein.In some embodiments, the guide RNA or the portion thereof that binds to the first RNA binding protein or the second RNA binding protein is non-covalently bound to the first RNA binding protein or the second RNA binding protein.

[0082] In some embodiments of the compositions of the present disclosure, the guide RNA or a portion thereof comprises or consists of between 10 and 100 nucleotides, including the end. In some embodiments, the spacer sequence of the present disclosure comprises or consists of between 10 and 30 nucleotides, including the end. In some embodiments, the spacer sequence of the present disclosure comprises or consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, the spacer sequence of the present disclosure comprises or consists of 20 nucleotides. In some embodiments, the spacer sequence of the present disclosure comprises or consists of 21 nucleotides. In some embodiments, the spacer sequence of the present disclosure comprises or consists of 26 nucleotides.

[0083] Guide molecules generally exist in various processing states. In one example, the unprocessed guide RNA is a 36nt DR followed by a 30-32nt spacer. The guide RNA is processed (shortened / modified) into a shorter "mature" form by Cas13d itself or other RNases. In some embodiments, the unprocessed guide sequence is about or at least about 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or more nucleotides (nt) in length. In some embodiments, the processed guide sequence is about 44-60 nt (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nt). In some embodiments, the unprocessed spacer is about 28-32 nt in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nt), while the mature (processed) spacer can be about 10-30 nt, 10-25 nt, 14-25 nt, 20-22 nt, or 14-30 nt (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nt). In some embodiments, the unprocessed DR is about 36 nt (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 nt), while the processed DR is about 30 nt (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nt). In some embodiments, the DR sequence is truncated, e.g., at the 5' end, by 1-10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, -10 nucleotides) and expressed as a mature pre-processed guide RNA.

[0084] In some embodiments of the compositions of the present disclosure, the guide RNA or portion thereof does not comprise a nuclear localization sequence (NLS).

[0085] In some embodiments of the compositions of the present disclosure, the guide RNA or a portion thereof comprises a sequence complementary to the protospacer flanking sequence (PFS). In some embodiments, the first RNA binding protein may comprise a sequence isolated or derived from a Cas13 protein, including those in which the guide RNA or a portion thereof comprises a sequence complementary to the PFS. In some embodiments, the first RNA binding protein may comprise a sequence encoding a Cas13 protein or an RNA binding portion thereof, including those in which the guide RNA or a portion thereof comprises a sequence complementary to the PFS. In some embodiments, the guide RNA or a portion thereof does not comprise a sequence complementary to the PFS.

[0086] In some embodiments of the compositions of the present disclosure, the vector comprising the guide RNA sequence of the present disclosure comprises a promoter sequence for driving the expression of the guide RNA. In some embodiments, the vector comprising the guide RNA sequence of the present disclosure comprises a promoter sequence for driving the expression of the guide RNA. In some embodiments, the promoter for driving the expression of the guide RNA is a constitutive promoter. In some embodiments, the promoter sequence is an inducible promoter. In some embodiments, the promoter is a sequence is a tissue-specific and / or cell type-specific promoter. In some embodiments, the promoter is a hybrid or recombinant promoter. In some embodiments, the promoter is a promoter that can express the guide RNA in a mammalian cell. In some embodiments, the promoter is a promoter that can express the guide RNA in a human cell. In some embodiments, the promoter is a promoter that can express the guide RNA and restrict the guide RNA to the nucleus of the cell. In some embodiments, the promoter is a human RNA polymerase promoter or a sequence isolated or derived from a sequence encoding a human RNA polymerase promoter. In some embodiments, the promoter is a U6 promoter or a sequence isolated or derived from a sequence encoding a U6 promoter. In some embodiments, the U6 promoter is a human U6 promoter. In some embodiments, the promoter is a human tRNA promoter or a sequence isolated or derived from a sequence encoding a human tRNA promoter. In some embodiments, the promoter is a human valine tRNA promoter or a sequence isolated or derived from a sequence encoding a human valine tRNA promoter.

[0087] In some embodiments of the composition of the present disclosure, the promoter for driving the expression of guide RNA further comprises a regulatory element.In some embodiments, the vector comprising the promoter sequence for driving the expression of guide RNA further comprises a regulatory element.In some embodiments, the regulatory element enhances the expression of guide RNA.Exemplary regulatory elements include, but are not limited to, enhancer elements, introns, exons, or combinations thereof.

[0088] In some embodiments of the composition of the present disclosure, the vector of the present disclosure comprises one or more of a sequence encoding a guide RNA, a promoter sequence for driving the expression of the guide RNA, and a sequence encoding a regulatory element. In some embodiments of the composition of the present disclosure, the vector further comprises a sequence encoding a fusion protein of the present disclosure. RNA-guided RNA-binding proteins

[0089] In some embodiments of the compositions of the present disclosure, the gRNA corresponds to a target RNA molecule and an RNA-guided RNA-binding protein. In some embodiments, the gRNA corresponds to an RNA-guided RNA-binding fusion protein, the fusion protein comprising a first and a second RNA-binding protein. In some embodiments, the first RNA-binding protein in the fusion protein is an inactivated RNA-binding protein, such as an inactivated Cas or a catalytically inactivated Cas protein. In some embodiments, along the sequence encoding the RNA-binding fusion protein, the sequence encoding the first RNA-binding protein is located 5' to the sequence encoding the second RNA-binding protein. In some embodiments, along the sequence encoding the fusion protein, the sequence encoding the first RNA-binding protein is located 3' to the sequence encoding the second RNA-binding protein.

[0090] In some embodiments of the compositions of the present disclosure, the sequence encoding the first RNA-binding protein comprises a sequence isolated or derived from a protein that can bind to an RNA molecule. In some embodiments, the sequence encoding the first RNA-binding protein comprises a sequence isolated or derived from a protein that can selectively bind to an RNA molecule and not bind to a DNA molecule, not bind to a mammalian DNA molecule, or not bind to any DNA molecule. In some embodiments, the sequence encoding the first RNA-binding protein comprises a sequence isolated or derived from a protein that can bind to an RNA molecule and induce breakage of the RNA molecule. In some embodiments, the sequence encoding the first RNA-binding protein comprises a sequence isolated or derived from a protein that can bind to an RNA molecule and induce breakage of the RNA molecule and not bind to a DNA molecule, not bind to a mammalian DNA molecule, or not bind to any DNA molecule. In some embodiments, the sequence encoding the first RNA binding protein comprises a sequence isolated from or derived from a protein that can bind to and induce destruction of an RNA molecule, but does not bind to and induce destruction of a DNA molecule, does not bind to and induce destruction of a mammalian DNA molecule, or does not bind to and induce destruction of any DNA molecule.

[0091] In some embodiments of the compositions of the present disclosure, the sequence encoding the RNA-binding protein guided by the first RNA comprises a sequence isolated or derived from a protein that does not have DNA nuclease activity.

[0092] In some embodiments of the composition of the present disclosure, the sequence encoding the RNA-guided RNA-binding protein disclosed herein comprises a sequence isolated or derived from a CRISPR Cas protein. In some embodiments, the CRISPR Cas protein is not a type II CRISPR Cas protein. In some embodiments, the CRISPR Cas protein is not a Cas9 protein.In some embodiments, the Cas9 protein is engineered to target RNA (RCas9). In some embodiments of the compositions of the present disclosure, the sequence encoding the RNA-guided RNA-binding protein comprises a type VI CRISPR Cas protein or a portion thereof. In some embodiments, the type VI CRISPR Cas protein comprises a Cas13 protein or a portion thereof. Exemplary Cas13 proteins of the present disclosure can be isolated or derived from any species, including, but not limited to, bacteria or archaea. Exemplary Cas13 proteins of the present disclosure include, but are not limited to, Leptotrichia wadei, Listeria seeligeri serovar 1 / 2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954), Lachnospiraceae bacterium, Clostridium aminophilum DSM 10710, Carnobacterium gallinarum DSM 4847, Paludibacter propionicigenes WB4, Listeria weihenstephanensis FSL R9-0317, Listeria weihenstephanensis FSL R9-0317, bacterium FSL M6-0635 (Listeria newyorkensis), Leptotrichia wadei F0279, Rhodobacter capsulatus SB 1003, Rhodobacter capsulatus The exemplary Cas13 protein of the present disclosure can be isolated or derived from any species, including Rhodobacter capsulatus DE442 and Corynebacterium ulcerans. The exemplary Cas13 protein of the present disclosure can be DNA nuclease inactivated. The exemplary Cas13 protein of the present disclosure includes, but is not limited to, Cas13a, Cas13b, Cas13c, Cas13d and their orthologues.Exemplary Cas13b proteins of the present disclosure include, but are not limited to, subtypes 1 and 2, referred to herein as Csx27 and Csx28, respectively. AAV viral vectors

[0093] "Viral vector" is defined as a recombinant virus or virus particle that contains a polynucleotide to be delivered to a host cell in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, etc. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, for example, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0094] "AAV virion" or "AAV virus particle" or "AAV virus vector" or "rAAV virus vector" or "AAV vector particle" or "AAV particle" refers to a virus particle that is composed of at least one AAV capsid protein and a polynucleotide rAAV vector enclosed in the capsid.Thus, the production of a rAAV virus vector necessarily includes the production of a rAAV vector, and therefore the vector is contained within the rAAV vector.

[0095] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or coat of a virus particle. Capsid functions to encapsidate, protect, transport and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed in the viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2 and VP3. The mixture of VP1, VP2 and VP3 contains 60 monomers arranged in a T=1 icosahedral symmetry in a ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3), as described in Sonntag F et al., (June 2010). "A viral assembly factor promotes AAV2 capsid formation in the nucleolus". Proceedings of the National Academy of Sciences of the United States of America. 107 (22): 10220-5, and Rabinowitz JE, Samulski RJ (December 2000). "Building a better vector: the manipulation of AAV virions". Virology. 278 (2): 301-8, each of which is incorporated herein by reference in its entirety.

[0096] The present disclosure provides an rAAV viral vector comprising: a) any of the rAAV vectors described herein; and b) an AAV capsid protein.

[0097] The AAV capsid protein can be any AAV capsid protein known in the art.In some embodiments, the AAV capsid protein is a modified AAV capsid protein.The AAV capsid protein can be AAV1 capsid protein, AAV2 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAV13 capsid protein, AAVPHP.B capsid protein, AAVrh74 capsid protein or AAVrh.10 capsid protein.The AAV capsid protein can be any modified AAV capsid protein of the present disclosure. Modified AAV capsid proteins

[0098] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or coat of a virus particle. Capsid functions to enclose, protect, transport and release the viral genome into the host cell. Capsid is generally composed of oligomeric structural subunits of proteins ("capsid protein"). As used herein, the term "encapsidated" means enclosed within the viral capsid. Modified AAV capsid proteins are provided herein that can be used to construct modified and / or chimeric AAV vectors or AAV capsids.

[0099] AAV capsids are generally composed of a total of 60 molecules of viral proteins (VPs) VP1, VP2 and VP3 in a ratio of about 1:1:10. VP1, VP2 and VP3 are encoded by the cap open reading frame and are generated by alternative splicing of mRNA and the use of alternative translation initiation codons. The VP3 sequence of about 524-544 amino acids (aa) is shared among all VPs, the VP2 sequence is approximately 57 aa longer than VP3 (about 580-601 aa), and the VP1 sequence is approximately 137 aa longer than VP2 (about 713-738 aa). The VP3 common region assembles the dodecahedral capsid. See Woerner et al. Nature Communications Vol. 12, Article number: 1642 (2021).

[0100] In some aspects, the modified AAV capsid proteins of the present disclosure are derived from any AAV serotype known in the art. The AAV capsid proteins can be derived from any AAV capsid protein known in the art. In some embodiments, the AAV capsid protein can be derived from AAV1 capsid protein, AAV2 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAV13 capsid protein, AAVPHP.B capsid protein, AAVrh8 capsid protein, AAVrh74 capsid protein, or AAV-TT (AAVv66) capsid protein, AAV PO1 capsid protein, AAVDJ, or AAVrh10 capsid protein. In some embodiments, the AAV capsid protein is an AAV-TT capsid protein. In some embodiments, the AAV capsid protein is an AAVrhlO capsid protein. In some embodiments, the modified AAV capsid protein of the present disclosure can be a chimeric AAV capsid protein derived from two or more AAV capsid proteins.

[0101] Modified AAV capsid protein sequences are disclosed herein. As used herein, "modified AAV capsid protein" or "modified capsid protein" refers to AAV capsid protein that is modified relative to wild-type AAV capsid protein sequence. Modified AAV capsid protein can include any one of capsid proteins VP1, VP2 or VP3. Modification of AAV capsid protein sequence can be any protein modification known in the art, including amino acid deletion, mutation, insertion or rearrangement. Modification of AAV capsid protein can be the formation of chimeric AAV capsid protein, where two or more AAV capsid protein regions are spliced ​​and combined with each other to form hybrid or chimeric AAV capsid protein. In some aspects, the modified AAV capsid protein that is composed of hybrid or chimeric AAV capsid protein comprises two or more AAV capsid protein regions, each of which has a unique serotype. In some embodiments, the hybrid capsid protein is a swap of variable domain loop regions of capsid proteins, hi some embodiments, the hybrid AAV capsid protein comprises variable region loops from two or more capsid sequences having different serotypes.

[0102] The modified AAV capsid proteins of the present disclosure can include a peptide insertion from any protein or peptide known in the art. In some aspects, the inserted peptide can be derived from a non-AAV capsid protein.

[0103] The modified AAV capsid proteins of the present disclosure can include any combination of modifications. As a non-limiting example, the AAV capsid proteins of the present disclosure can be chimeric, as well as contain at least one of an amino acid deletion, mutation, insertion, or rearrangement.

[0104] The modified AAV capsid proteins of the present disclosure can be used to form AAV capsids with improved properties, including increased transduction in certain tissue types (i.e., "on-target transduction") and / or reduced transduction in undesirable tissue types (i.e., "off-target transduction"). In some embodiments, muscle tissue-specific transduction is observed. In some embodiments, ocular tissue-specific transduction is observed. In some embodiments, neuronal or nervous tissue-specific transduction is observed. In some embodiments, transduction in the liver is reduced or eliminated. In some embodiments, AAV capsids comprising modified AAV capsid proteins have reduced transduction in non-targeted tissues or cell types. In some embodiments, non-targeted tissues include the liver, lung, kidney, brain, spleen, intestine, spinal cord, or reproductive organs.

[0105] Without wishing to be bound by theory, AAV vectors comprising the modified AAV capsid proteins of the present disclosure may have reduced reactivity to pre-existing neutralizing antibodies in human subjects due to capsid modifications that generate unique binding epitopes not observed in commonly used AAV capsid serotypes. Peptide insertion in the AAV capsid protein

[0106] Peptides or amino acids can be inserted into any region of the AAV capsid protein. Insertions can be made at the N-terminus or C-terminus of the protein. In some embodiments, insertions can be made in any variable region (VR) of the capsid protein, including VR1 (VRI), VR2 (VRII), VR3 (VRIII), VR4 (VRIV), VR5 (VRV), VR6 (VRVI), VR7 (VRVII), VR8 (VRVIII), or VR9 (VRX).

[0107] In some aspects, the modified AAV capsid protein of the present disclosure comprises a modified variable region. In some aspects, the modified AAV capsid protein of the present disclosure comprises a modified VR VIII region. In some aspects, the modification of the AAV capsid protein is an insertion into VR VIII. Peptide Insertion Sequence

[0108] The peptide sequence was selected to enhance transduction in a tissue-specific manner. Any sequence that enhances tissue-specific transduction is contemplated. In some aspects, muscle tissue-specific transduction is observed. In some aspects, ocular tissue-specific transduction is observed. In some aspects, neuronal or nervous tissue-specific transduction is observed. The inserted peptide can target tissue-specific receptors, resulting in increased transduction in said tissue.

[0109] In some embodiments, the modified AAV capsid protein contains a peptide insertion that targets the insulin receptor (INSR). AAV vectors designed to target the INSR have been shown to enhance intramuscular transduction (see Jackson et al. Molecular Therapy Methods & Clinical Development, 2020, 19, 11, 496-506, which is incorporated herein by reference in its entirety). In some embodiments, the INSR targeting peptide inserted is an insulin mimetic peptide called S519. In some embodiments, the S519 peptide may comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence SLEEEWAQVECEVYGRGCPSGSLDESFYDWFERQL (SEQ ID NO: 301).

[0110] In some embodiments, the modified AAV capsid protein contains a peptide insertion that targets muscle specific kinase (MUSK). MUSK expression in the liver has been shown to be very low or absent. It is known that the acetylcholinesterase collagenoplastic tail peptide (ColQ) binds to and targets MUSK. In some embodiments, the inserted MUSK targeting peptide is the C-terminal portion of ColQ (ColQ CTD). In some embodiments, the ColQ CTD peptide has the amino acid sequence [ka] It may comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to

[0111] In some embodiments, the modified AAV capsid protein comprises an integrin targeting peptide insertion. In some embodiments, the integrin targeting peptide comprises an RGD motif. RGD sequences are known in the art and include the motif RGDXXXX, which can be inserted into AAV viral vectors for targeting by integrin class receptors, for example. For examples of RGD sequences that can be used in the modified AAV capsids described herein, see, for example, Michelfelder et al., PLoS One. 2009; 4(4): e5122, which is incorporated herein by reference in its entirety. It has been shown that RGD motif peptide insertion into VR8 of AAV9 increases mouse muscle transduction (see Weinmann et al. Nature Communications, 11:5432, which is incorporated herein by reference in its entirety). In some embodiments, the RGD peptide comprises partial sequence Y or F amino acid to produce RGDY or RGDF motif. RGDY or RGDF motifs have been demonstrated to result in enhanced muscle transduction in non-human primates (NHPs) (see Tabeboldbar et al. Cell, 184, 19, 2021, 4919-4938, the entire contents of which are incorporated herein by reference). In some embodiments, the RGD sequence comprises RGDLGLS (SEQ ID NO: 303). In some embodiments, the RGD sequence comprises RGDLSTP (SEQ ID NO: 304), SNSRGDYNSL (SEQ ID NO: 305), ENRRGDFNNT (SEQ ID NO: 306), SRGDYNSL (SEQ ID NO: 307), RGDYNSL (SEQ ID NO: 308), RGDLST (SEQ ID NO: 309) or RGDYVGL (SEQ ID NO: 310).

[0112] In some embodiments, the RGD sequence of the present disclosure can be inserted into a scaffold (Figure 8). In some embodiments, the RGD sequence of the present disclosure comprises a linker at one or more of the N-terminus and C-terminus forming a linker scaffold. In some embodiments, the linker scaffold comprises a flexible linker, for example, GGGS (SEQ ID NO: 311). In some embodiments, the linker scaffold comprises a rigid scaffold, for example, a VHH, GP2, cyclic peptide, or knottin scaffold.

[0113] In some embodiments, the RGD sequence is a variable domain (VHH) RGD peptide of a camelid heavy chain only antibody, e.g., the amino acid sequence [ka] (RGD motif underlined), comprising, consisting essentially of or consisting of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to VHH RGD peptide.

[0114] In some embodiments, the RGD peptide insertion comprises a glycoprotein 2 (GP2) peptide scaffold insertion. In some embodiments, the GP2 scaffold peptide insertion comprises the amino acid sequence KFWATVGRGDLSTPFEVPVYAETLDEALELAENRRGDFNNTVTRVRP (SEQ ID NO: 317) or GGGGSGGGGSKFWATVGRGDLSTPFEVPVYAETLDEALELAENRRGDFNNTVTRVRPGGGGS (SEQ ID NO: 318).

[0115] In some embodiments, the RGD peptide insertion may comprise a knottin scaffold peptide insertion. In some embodiments, the knottin scaffold peptide insertion comprises the amino acid sequence NSRGDYNSLSCSQDSDCLAGCVCGPNGFC (SEQ ID NO: 319) or GGGGSGGGGSGCSNSRGDYNSLSCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO: 320).

[0116] In some embodiments, the RGD peptide insertion may comprise a cyclic peptide scaffold RGD peptide insertion. In some embodiments, the cyclic peptide scaffold RGD peptide insertion comprises the amino acid sequence ACRGDYNSLCRGDLSTC (SEQ ID NO: 321) or GGGGACRGDYNSLCRGDLSTCGGGGS (SEQ ID NO: 322).

[0117] The scaffold can be used to insert any peptide known in the art. Thus, VHH, GP2, cyclic peptides, knottins, or flexible linkers can be used to scaffold or flank any peptide for insertion into the modified AAV capsid protein of the present disclosure.

[0118] The inserted peptide may be flanked at the N- or C-terminus by a flexible linker peptide of any length. In some embodiments, a flexible linker, such as GGGS (SEQ ID NO: 311), is used. In some embodiments, the flexible GGGS linker may be repeated multiple times to form a longer linker sequence. In some embodiments, the linker is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the linker sequence comprises GGGGS (SEQ ID NO: 311), GGGGSGGGGS (SEQ ID NO: 312), GGGGSGGGGSGGGGGS (SEQ ID NO: 313), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 314), or GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 315). Point mutations

[0119] Additionally or alternatively, modified AAV capsid proteins may contain amino acid mutations that result in increased transduction in desired tissue types. In some aspects, modified AAV capsid proteins may contain amino acid mutations that result in reduced transduction in specific tissue types. In some aspects, modified AAV capsid proteins may contain amino acid mutations that result in reduced liver tissue transduction.

[0120] In some embodiments, the modified AAV capsid protein comprises an amino acid sequence provided herein (e.g., a sequence selected from Table 1) with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid changes. The amino acid changes can be the substitution of an amino acid with any other amino acid, including natural amino acids and non-natural or modified amino acids. In some aspects, the mutation is a conservative amino acid mutation. A conservative amino acid substitution is the replacement of an amino acid in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue; or the substitution of one charged or polar residue with another charged or polar residue, such as the substitution of lysine with arginine, the substitution of aspartic acid with glutamic acid, the substitution of asparagine with glutamine, etc. In some embodiments, conservative amino acid substitutions are selected from alanine to serine; asparagine to glutamine or histidine, aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine.

[0121] In some embodiments, a mutation at position 503 of AAVrh8 results in reduced liver transduction. In some embodiments, a substitution of alanine with tryptophan at position 503 of AAVrh8 results in reduced liver transduction. In some embodiments, a mutation at position 505 of AAVrh74 results in reduced liver transduction. In some embodiments, a substitution of alanine with tryptophan at position 55 of AAVrh74 results in reduced liver transduction. In some embodiments, a mutation at position 498 of AAV9 capsid protein results in reduced liver transduction. In some embodiments, the AAV9 point mutation comprises an N498 mutation. In some embodiments, the AAV9 point mutation comprises an N498I mutation. In some embodiments, a mutation at position 602 of AAV capsid protein results in reduced liver transduction.

[0122] Robust liver detargeting in NHPs is suggested by mutating the following residues in AAV9 (F501I, G505R, Y706C). In some embodiments, the liver detargeting mutations in AAV9 include at least one of F501I, G505R, Y706C. The equivalent mutations in certain vectors of the present disclosure include F503I, G507R, and Y708C in LBV30 to generate LBV92. In some embodiments, the AAV Rh74 liver detargeting mutations include any combination of mutations of residues F503, G507, Y707, and / or Y708 of AAV Rh74. For some embodiments, these mutations include F503I, G507R, Y707C, and / or Y708C of AAV Rh74.

[0123] In some embodiments, the point mutations of the present disclosure are numbered relative to the wild-type AAV capsid protein sequence.The wild-type AAV capsid protein sequence is disclosed in Table XXX.In some embodiments, the modified AAV capsid protein of the present disclosure comprises insertions and / or deletions or variable region exchanges that can change the length of the capsid protein sequence.It should be understood that the point mutations referred to in Table X1 are relative to the wild-type or unmodified AAV capsid protein sequence. Modified AAV capsid sequences

[0124] The present disclosure provides modified AAV capsid protein amino acid sequences. The present disclosure further provides nucleic acid sequences encoding the modified AAV capsid proteins of the present disclosure.

[0125] The modified AAV capsid proteins of the present disclosure are shown in Table 1. Table 1 lists the identities of assembled AAV capsids, including VP1, VP2 and VP3 capsid proteins, including the modified AAV capsid proteins disclosed herein. Table 1 lists the serotypes of the capsid proteins as well as any modifications made to the capsid sequence. Table 1: AAV capsid protein sequences of the present disclosure [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0126] In some aspects, the modified AAV capsid VP1 proteins provided herein comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VP1 sequence set forth in Table 1.

[0127] In some embodiments, the modified AAV capsid VP2 / 3 proteins provided herein comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (or any percentage in between) identical to the VP2 / VP3 sequences shown in Table 1.

[0128] In some embodiments, the modified AAV capsid VP1 protein provided herein is encoded by a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VP1 sequence shown in Table 1.

[0129] In some embodiments, the modified AAV capsid VP2 / 3 proteins provided herein are encoded by a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP2 / VP3 sequences shown in Table 1.

[0130] In some aspects, the muscle-targeted modified AAV capsid VP1 proteins provided herein comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VP1 sequence set forth in Table 1.

[0131] In some aspects, the muscle-targeted modified AAV capsid VP2 / 3 proteins provided herein comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP2 / VP3 sequences set forth in Table 1.

[0132] In some embodiments, the muscle-targeted modified AAV capsid VP1 protein provided herein is encoded by a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VP1 sequence shown in Table 1.

[0133] In some embodiments, the muscle-targeting modified AAV capsid VP2 / 3 proteins provided herein are encoded by a polynucleotide that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP2 / VP3 sequences shown in Table 1.

[0134] In another aspect, provided herein are modified AAV capsid proteins that are useful for in vitro transduction.

[0135] In some embodiments, the modified VP1 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 53. In some embodiments, SEQ ID NO: 53 is an AAV9 VP1 capsid protein comprising a VHH RGD peptide insertion. In some embodiments, the modified VP1 capsid protein set forth in SEQ ID NO: 53 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 151.

[0136] In some embodiments, the modified VP2 / VP3 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 102. In some embodiments, SEQ ID NO: 102 is an AAV9 VP2 / 3 capsid protein. In some embodiments, the VP2 / VP3 capsid protein set forth in SEQ ID NO: 102 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 199.

[0137] In some embodiments, the modified VP1 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 75. In some embodiments, SEQ ID NO: 75 is a Rh74 VP1 capsid protein that includes an mColQ peptide insertion and the mutations F503I, G507R, and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein). In some embodiments, the modified VP1 capsid protein set forth in SEQ ID NO: 75 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 173.

[0138] In some embodiments, the modified VP2 / VP3 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 124. In some embodiments, SEQ ID NO: 124 is a modified Rh74 VP2 / 3 capsid protein that includes an RGD peptide insertion and the mutations F503I, G507R, and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein). In some embodiments, the modified VP2 / VP3 capsid protein set forth in SEQ ID NO: 124 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 221.

[0139] In some embodiments, the modified VP1 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 245. In some embodiments, SEQ ID NO: 245 is a Rh74 VP1 capsid protein comprising a VHH RGD peptide insertion and the mutations F503I, G507R, and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein). In some embodiments, the modified VP1 capsid protein set forth in SEQ ID NO: 245 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 270.

[0140] In some embodiments, the modified VP2 / VP3 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 258. In some embodiments, SEQ ID NO: 258 is a modified Rh74 VP2 / 3 capsid protein that includes an RGD peptide insertion and the mutations F503I, G507R, and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein). In some embodiments, the modified VP2 / VP3 capsid protein set forth in SEQ ID NO: 258 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 283.

[0141] In some embodiments, the modified VP1 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 249. In some embodiments, SEQ ID NO: 249 is an AAV9 VP1 capsid protein comprising a VHH RGD peptide insertion and the mutation N498I (amino acid numbering relative to the wild-type AAV9 capsid protein). In some embodiments, the modified VP1 capsid protein set forth in SEQ ID NO: 249 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 274.

[0142] In some embodiments, the modified VP2 / VP3 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 262. In some embodiments, SEQ ID NO: 262 is a modified AAV9 VP2 / 3 capsid protein that includes an RGD peptide insertion and the mutation N498L (amino acid numbering relative to the wild-type AAV9 capsid protein). In some embodiments, the modified VP2 / VP3 capsid protein set forth in SEQ ID NO: 262 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 287.

[0143] In some embodiments, the modified VP1 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 255. In some embodiments, SEQ ID NO: 255 is an AAV9 VP1 capsid protein comprising a VHH RGD peptide insertion. In some embodiments, the modified VP1 capsid protein set forth in SEQ ID NO: 255 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 280.

[0144] In some embodiments, the modified VP2 / VP3 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 267. In some embodiments, SEQ ID NO: 267 is a modified AAV9 VP2 / 3 capsid protein that includes an RGD peptide insertion. In some embodiments, the modified VP2 / VP3 capsid protein set forth in SEQ ID NO: 267 is encoded by a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 292. Modified AAV capsids

[0145] Modified AAV capsids can be formed by forming AAV capsids using the modified AAV capsid proteins of the present disclosure.Modified AAV capsid proteins provided herein can be used to construct modified, chimeric and / or hybrid AAV capsids.AAV capsid provided herein can include any combination of VP1, VP2 and VP3 sequences, or VP1 and VP2 / VP3 proteins.

[0146] In some embodiments, the modified AAV capsids provided herein comprise a wild-type or modified VP1 capsid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP1 sequence set forth in Table 1 and the SEQ ID NOs referenced therein. In some embodiments, the modified AAV capsids provided herein comprise a wild-type or modified VP2 / VP3 capsid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP2 / VP3 sequence set forth in Table 1 and the SEQ ID NOs referenced therein. In some embodiments, a modified AAV capsid of the disclosure comprises a VP1 capsid protein selected from the sequences listed in Table 1, and a VP2 / VP3 capsid protein selected from the sequences listed in Table 1.

[0147] In some embodiments, the modified AAV capsid comprises a VP1 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO:53, and a VP2 / VP3 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO:102. In some embodiments, the AAV capsid having the sequences is referred to as LBV55. LBV55 comprises an AAV9 VP1 capsid protein containing a VHH RGD peptide insert and an AAV9 VP2 / VP3 capsid protein.

[0148] In some embodiments, the modified AAV capsid comprises a VP1 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 75, and a VP2 / VP3 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 124. In some embodiments, the AAV capsid having said sequences is referred to as LBV93. LBV93 contains an Rh74 VP1 capsid protein containing an mColQ peptide insertion and the mutations F503I, G507R, and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein), and an Rh74 VP2 / VP3 capsid protein containing an RGD peptide insertion and the mutations F503I, G507R, and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein).

[0149] In some embodiments, the modified AAV capsid comprises a VP1 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 245, and a VP2 / VP3 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 258. In some embodiments, the AAV capsid having said sequences is designated LBV110. LBV110 contains an Rh74 VP1 capsid protein containing a VHH RGD peptide insertion and the mutations F503I, G507R and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein), and an Rh74 VP2 / VP3 capsid protein containing an RGD peptide insertion and the mutations F503I, G507R and Y707C (amino acid numbering relative to the wild-type Rh74 capsid protein).

[0150] In some embodiments, the modified AAV capsid comprises a VP1 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 249, and a VP2 / VP3 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 262. In some embodiments, the AAV capsid having said sequences is designated LBV114. LBV114 contains an AAV9 VP1 capsid protein containing a VHH RGD peptide insertion and the mutation N498I (amino acid numbering relative to the wild-type AAV9 capsid protein), and an AAV9 VP2 / VP3 capsid protein containing an RGD peptide insertion and the mutation N498L (amino acid numbering relative to the wild-type AAV9 capsid protein).

[0151] In some embodiments, the modified AAV capsid comprises a VP1 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 255, and a VP2 / VP3 capsid protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 267. In some embodiments, the AAV capsid having the sequences is referred to as LBV121. LBV121 comprises an AAV9 VP1 capsid protein that includes a VHH RGD peptide insert, and an AAV9 VP2 / VP3 capsid protein that includes an RGD peptide insert.

[0152] The modified AAV capsid of the present disclosure shows improved transduction efficiency compared to parent or wild-type AAV capsid.In some aspects, the improved transduction is in muscle tissue.In some aspects, the muscle tissue is skeletal muscle, smooth muscle or cardiac muscle.In some embodiments, the transduction efficiency of the modified muscle-targeted AAV capsid provided herein in muscle cells is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to parent AAV capsid, as determined by immunofluorescence. In some embodiments, the transduction efficiency in muscle cells of the modified muscle-targeted AAV capsids provided herein is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the parent AAV capsid, as determined by immunofluorescence.

[0153] Viral vectors and pharmaceutical compositions

[0154] The AAV capsid of the present disclosure can be used in any suitable AAV viral vector. The AAV capsid encloses a small single-stranded DNA genome of approximately 4.8 kilobases (kb). The genome of AAV contains three genes Rep (replication), Cap (capsid) and aap (assembly) that are flanked by terminal inverted repeats (ITRs) that are required for genome replication and packaging. See Naso et al., BioDrugs. 2017; 31(4): 317-334. Recombinant AAV vectors lack viral genes and instead contain a transgene that is flanked by two viral ITRs.

[0155] Thus, in another aspect, provided herein is an AAV viral vector comprising the modified AAV capsid provided herein. In some embodiments, the AAV viral vector comprises a transgene.

[0156] Also provided herein is a pharmaceutical composition comprising an AAV viral vector comprising the modified AAV capsid provided herein and a pharma- ceutically acceptable carrier.The pharmaceutical composition can be formulated for any suitable route of administration, including, for example, intravenous, intrathecal, intracranial or intraocular administration.The pharmaceutical composition for use disclosed herein can comprise a polynucleotide encoding a protein(s) or a protein(s) optionally contained within an AAV, which is also optionally immuno-orthogonal, in combination with one or more pharma- cetically or physiologically acceptable carriers, diluents or excipients.Such compositions can comprise a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure can be formulated for administration routes, such as, for example, oral, enteral, topical, transdermal, intranasal and / or inhalation administration routes, as well as for administration routes, such as, for example, intravenous, intramuscular, subpial, intrathecal, intraparenchymal, intrathecal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular and / or parenteral administration, by injection or infusion. In certain embodiments, the compositions of the present disclosure are formulated for intracerebral or intrastriatal administration. How to use

[0157] The present disclosure provides a method of delivering a transgene or NOI to a tissue of interest in a subject, the method comprising administering an AAV viral vector comprising a modified AAV capsid protein of the present disclosure. In one embodiment, the present disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule, the method comprising contacting an AAV viral vector of the present disclosure and the RNA molecule under conditions suitable for binding of one or more of a guide RNA or an RNA binding protein or a fusion protein (or a portion thereof) to the RNA molecule.

[0158] The present disclosure provides a method for modifying the level of expression of an RNA molecule of the present disclosure or the expression of a protein encoded by the RNA molecule, comprising contacting an AAV viral vector of the present disclosure with a cell comprising the RNA molecule under conditions suitable for binding of one or more of a guide RNA or an RNA binding protein or a fusion protein (or a portion thereof) to the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In some embodiments, the AAV viral vector of the present disclosure comprises a guide RNA of the present disclosure and an RNA binding protein or a fusion protein of the present disclosure.

[0159] The present disclosure provides a method of modifying the activity of a protein encoded by an RNA molecule, the method comprising contacting an AAV viral vector of the present disclosure with a cell containing the RNA molecule under conditions suitable for binding of one or more of a guide RNA or an RNA binding protein or a fusion protein (or a portion thereof) to the RNA molecule.

[0160] The present disclosure provides a method for altering the level of expression of an RNA molecule of the present disclosure or expression of a protein encoded by the RNA molecule, comprising contacting an AAV viral vector of the present disclosure with an RNA molecule under conditions suitable for RNA nuclease activity of an RNA binding protein or fusion protein to induce destruction of the RNA molecule.

[0161] The present disclosure provides a method for altering the activity of a protein encoded by an RNA molecule, the method comprising contacting an AAV viral vector of the present disclosure with an RNA molecule under conditions suitable for RNA nuclease activity of an RNA-binding protein or fusion protein to induce destruction of the RNA molecule.

[0162] The present disclosure provides a method for modifying the level of expression of an RNA molecule of the present disclosure or the expression of a protein encoded by the RNA molecule, comprising contacting an AAV viral vector of the present disclosure with a cell comprising the RNA molecule under conditions suitable for RNA nuclease activity, where the RNA binding protein or fusion protein induces the destruction of the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In some embodiments, the AAV viral vector of the present disclosure comprises a guide RNA of the present disclosure and an RNA binding fusion protein of the present disclosure.

[0163] The present disclosure provides a method for modifying the activity of a protein encoded by an RNA molecule, comprising contacting an AAV viral vector of the present disclosure with a cell comprising the RNA molecule under conditions suitable for RNA nuclease activity, where the RNA binding protein or fusion protein induces the destruction of the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In some embodiments, the AAV viral vector of the present disclosure comprises a nucleic acid sequence encoding a guide RNA or single guide RNA of the present disclosure and an RNA binding protein or fusion protein of the present disclosure.

[0164] The present disclosure provides a method for treating a subject with disease or disorder, comprising administering to the subject a therapeutically effective amount of the AAV viral vector or pharmaceutical composition of the present disclosure.In some embodiments, the disease or disorder is a muscle and / or neuromuscular disease or disorder.In some embodiments, the muscle and / or neuromuscular disorder is a muscular dystrophy or myotonic dystrophy.

[0165] The present disclosure provides a method of treating a disease in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of an AAV viral vector or pharmaceutical composition of the present disclosure, wherein the AAV viral vector or pharmaceutical composition comprises a vector comprising a guide RNA of the present disclosure and a nucleic acid sequence encoding an RNA-binding protein or an RNA-binding protein fusion protein of the present disclosure, and wherein the AAV viral vector or pharmaceutical composition modifies, reduces, disrupts, lowers, or eliminates the level of expression of a toxic repeat RNA (compared to the level of expression of the toxic repeat RNA treated with a non-targeting (NT) control or compared to no treatment). In another embodiment, the level of reduction is 1-fold or more. In another embodiment, the level of reduction is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. In another embodiment, the level of reduction is 10-fold or more. In another embodiment, the level of reduction is between 10-fold and 20-fold. In another embodiment, the level of reduction is 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold. In another embodiment, the gene therapy composition disclosed herein, when administered to a patient, results in 20%-100% degradation of the toxic repeat RNA. In one embodiment, the % disappearance of the toxic repeat RNA is 20-99%, 25%-99%, 50%-99%, 80%-99%, 90%-99%, or 95%-99%. In one embodiment, the % disappearance is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In another embodiment, the % disappearance is complete or 100% disappearance of the toxic repeat RNA.

[0166] In some embodiments of the method of the present disclosure, the subject of the present disclosure is diagnosed with the disease to be treated.In some embodiments, the subject of the present disclosure shows at least one sign or symptom of the disorder or disease to be treated.In some embodiments, the subject of the present disclosure shows at least one sign or symptom of the disease.

[0167] In some embodiments of the method of the present disclosure, the subject of the present disclosure is female.In some embodiments of the method of the present disclosure, the subject of the present disclosure is male.In some embodiments, the subject of the present disclosure has two chromosomes XX or XY.In some embodiments, the subject of the present disclosure has two chromosomes XX or XY and a third chromosome, either X or Y.

[0168] In some embodiments of the method of the present disclosure, the subject of the present disclosure is a newborn, an infant, a child, an adult, an elderly adult, or an older adult.In some embodiments of the method of the present disclosure, the subject of the present disclosure is at least 1 day old, 2 days old, 3 days old, 4 days old, 5 days old, 6 days old, 7 days old, 8 days old, 9 days old, 10 days old, 11 days old, 12 days old, 13 days old, 14 days old, 15 days old, 16 days old, 17 days old, 18 days old, 19 days old, 20 days old, 21 days old, 22 days old, 23 days old, 24 days old, 25 days old, 26 days old, 27 days old, 28 days old, 29 days old, 30 days old, or 31 days old. In some embodiments of the methods of the present disclosure, the subject of the present disclosure is at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months old. In some embodiments of the methods of the present disclosure, the subject of the present disclosure is at least 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 years old, 10 years old, 15 years old, 20 years old, 25 years old, 30 years old, 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old, 95 years old, 100 years old, or any age or partial age therebetween.

[0169] In some embodiments of the methods of the present disclosure, the subject of the present disclosure is a mammal. In some embodiments, the subject of the present disclosure is a non-human mammal.

[0170] In some embodiments of the methods of the present disclosure, the subject of the present disclosure is a human.

[0171] In some embodiments of the method of the present disclosure, the therapeutically effective amount comprises a single dose of the composition of the present disclosure.In some embodiments, the therapeutically effective amount comprises at least one dose of the composition of the present disclosure.In some embodiments, the therapeutically effective amount comprises one or more doses of the composition of the present disclosure.

[0172] In some embodiments of the methods of the present disclosure, a therapeutically effective amount is one that eliminates a sign or symptom of a disease or disorder, hi some embodiments, a therapeutically effective amount is one that reduces the severity of a sign or symptom of a disease or disorder.

[0173] In some embodiments of the methods of the present disclosure, a therapeutically effective amount is one that eliminates the disease or disorder.

[0174] In some embodiments of the disclosed methods, the therapeutically effective amount prevents the onset of a disease or disorder. In some embodiments, the therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, the therapeutically effective amount reduces the severity of a sign or symptom of a disease or disorder. In some embodiments, the therapeutically effective amount improves the prognosis of a subject.

[0175] In some embodiments of the method of the present disclosure, the composition of the present disclosure is administered to the subject via intracerebral administration. In some embodiments, the composition of the present disclosure is administered to the subject via intrastriatal route. In some embodiments, the composition of the present disclosure is administered to the subject by stereotactic injection or infusion. In some embodiments, the composition is administered to the brain. In some embodiments of the method of the present disclosure, the composition of the present disclosure is administered to the subject locally.

[0176] In some embodiments, the compositions disclosed herein are formulated as pharmaceutical compositions.Simply put, the pharmaceutical compositions for use disclosed herein can comprise a polynucleotide encoding a protein(s) or a protein(s) optionally contained in AAV, which is optionally also immuno-orthogonal, in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents or excipients.Such compositions can comprise a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrate, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid, such as glycine; antioxidant; chelating agent, such as EDTA or glutathione; adjuvant (e.g., aluminum hydroxide); and preservative. The compositions of the present disclosure can be formulated for routes of administration such as, for example, oral, enteral, topical, transdermal, intranasal and / or inhalation routes of administration; as well as for routes of administration such as, for example, intravenous, intrathecal, intracerebral, intravenous, intranasal, intratracheal, intraaural, intraocular or periocular, oral, rectal, transmucosal, by inhalation, transdermal, parenteral, subcutaneous, intradermal, intramuscular, intracisternal, intraneuronal, intrapleural, topical, intralymphatic, intracisternal, etc., by injection or infusion; such introduction can also be intra-arterial, intracardiac, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intraarticular, intra- ...

[0177] In some embodiments, after delivery of the AAV viral vector comprising modified AAV capsid protein, enhanced transduction occurs in muscle tissue.In some embodiments, after subretinal delivery of the AAV vector comprising modified AAV capsid protein compared to the AAV capsid comprising unmodified, wild-type or parent AAV capsid protein, enhanced transduction occurs in ocular tissue.In some embodiments, after delivery of the AAV vector comprising modified AAV capsid protein compared to the AAV capsid comprising unmodified, wild-type or parent AAV capsid protein, enhanced transduction occurs in neural tissue.In some embodiments, after systemic delivery of the AAV vector comprising modified AAV capsid protein compared to the AAV capsid comprising unmodified, wild-type or parent AAV capsid protein, reduced liver or hepatocyte transduction occurs, or no liver or hepatocyte transduction occurs. In some embodiments, after systemic delivery of AAV vectors comprising modified AAV capsids compared to AAV capsids comprising unmodified, wild-type or parent AAV capsid proteins, neutralizing antibody binding is reduced or does not occur.In some embodiments, neutralizing antibody titer is minimized after delivery of AAV vectors comprising modified AAV capsids compared to AAV capsids comprising unmodified, wild-type or parent AAV capsid proteins.In some embodiments, neutralizing antibody titer is reduced compared to delivery of AAV vectors comprising unmodified AAV capsids compared to AAV capsids comprising unmodified, wild-type or parent AAV capsid proteins. cell

[0178] In some embodiments of the compositions and methods of the present disclosure, the cells of the present disclosure are prokaryotic cells.

[0179] In some embodiments of the compositions and methods of the present disclosure, the cells of the present disclosure are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are bovine, murine, feline, equine, porcine, canine, simian, or human cells. In some embodiments, the cells are non-human mammalian cells, such as non-human primate cells.

[0180] In some embodiments, the cells of the present disclosure are somatic cells. In some embodiments, the cells of the present disclosure are germline cells. In some embodiments, the germline cells of the present disclosure are not human cells.

[0181] In some embodiments of the compositions and methods of the present disclosure, the cell of the present disclosure is a stem cell. In some embodiments, the cell of the present disclosure is an embryonic stem cell. In some embodiments, the embryonic stem cell of the present disclosure is not a human cell. In some embodiments, the cell of the present disclosure is a multipotent stem cell or a pluripotent stem cell. In some embodiments, the cell of the present disclosure is an adult stem cell. In some embodiments, the cell of the present disclosure is an induced pluripotent stem cell (iPSC). In some embodiments, the cell of the present disclosure is a hematopoietic stem cell (HSC).

[0182] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are muscle cells. In some embodiments, the muscle cells of the present disclosure are myoblasts or myocytes. In some embodiments, the muscle cells of the present disclosure are cardiomyocytes, skeletal muscle cells or smooth muscle cells. In some embodiments, the muscle cells of the present disclosure are striated cells. In one embodiment, the patient's cell or cells treated with the compositions disclosed herein include, without limitation, skeletal muscle (developing and mature muscle fibers and satellite cells), neuromuscular junctions, cardiomyocytes, smooth muscle cells, peripheral nervous system (neurons), peripheral motor neurons, and / or sensory neurons.

[0183] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are epithelial cells. In some embodiments, the epithelial cells of the present disclosure form fibroblasts, squamous epithelial cells, cuboidal epithelial cells, columnar epithelial cells, stratified epithelial cells, pseudostratified columnar epithelial cells or transitional epithelial cells. In some embodiments, the epithelial cells of the present disclosure form glands, including but not limited to the pineal gland, thymus, pituitary gland, thyroid gland, adrenal gland, apocrine gland, holocrine gland, part-secretory gland, serous gland, mucous gland and sebaceous gland. In some embodiments, the epithelial cells of the present disclosure are in contact with the outer surface of an organ, including but not limited to the lung, spleen, stomach, pancreas, bladder, intestine, kidney, gallbladder, liver, larynx or pharynx. In some embodiments, the epithelial cells of the present disclosure are in contact with the outer surface of a blood vessel or vein.

[0184] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are primary cells.

[0185] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are cultured cells.

[0186] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are in vivo, in vitro, ex vivo, or in situ. In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are autologous or allogeneic cells. EXAMPLES

[0187] The examples provided in this section are for illustrative purposes only and are not intended to limit the invention. Example 1 VP1-specific presentation of targeting peptides

[0188] The use of targeting peptides can increase transfection efficiency in certain tissues or cell types, but the way in which the peptide is presented by the AAV capsid influences the effect it has.

[0189] Figure 5 shows immunofluorescence images of HEK293 cells transfected with AAV Rh74 particles containing peptide insertions. Both eAAV Rh74 and LBV28 contain the insulin receptor targeting peptide, S519. eAAV Rh74 insertion sites VR8 for VP1, VP2 and VP3 at mutant 1-10 to wild type protein. LBV28 has the peptide specifically inserted into VR8 VP1 but not into VP2 or VP3. These data indicate that VP1-specific insertion of LBV28 results in greater efficacy of targeting peptides. Without wishing to be bound by theory, we hypothesize that VP1-specific insertion allows peptides to be presented more freely, resulting in greater efficacy, while on the other hand, problems with capsid assembly limit the use of linkers when inserting peptides into all VP1-3 proteins.

[0190] Example 2 Generation and testing of novel muscle-targeted AAV capsids

[0191] Novel and improved muscle-targeted capsid proteins were rationally designed from AAV9, AAVRh74 and AAVpo1. Rationally targeted capsid mutations and insertions were cloned and GFP and luciferase reporter viruses were produced using HEK293 cells and purification by iodixanol gradient ultracentrifugation. Coomassie staining was used to verify that the preparations were free of protein contamination. Endotoxin levels should be less than 1.5EU / ml. Titers were measured by ITR qPCR and engineered capsids had to be produced at 25% of the parental capsid to proceed to in vitro studies. C2C12 myoblast cells were used to evaluate the potential muscle targeting of engineered capsids. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS and transduction was visually assessed by GFP signal (both the percentage of GFP+ cells and GFP expression levels) using a fluorescent microscope. Briefly, C2C12 cells were seeded at a density of 5E4 cells per well in 48-well plates on day 1. On day 3, cells were transduced with AAV at a multiplicity of infection ((MOI); number of delivered viral genomes divided by total number of cells) of 1E5 and 1E6 based on 1E5 cells per well. GFP signal was monitored from days 4 to 10.

[0192] AAV capsids with modified AAV capsid proteins containing RGD peptide insertions were evaluated. LBV54 contains a flexible linker RGD peptide insertion in AAV9 VR VIII (VR8). LBV55 contains a VHH RGD peptide insertion in AAV9 VR VIII (VR8). LBV56 contains a GP2 RGD peptide insertion in AAV9 VR VIII (VR8). LBV57 contains a cyclic peptide RGD peptide insertion in AAV9 VR VIII (VR8). LBV58 contains a knottin RGD peptide insertion in AAV9 VR VIII (VR8) (Figure 8). AAV capsids with modified AAV capsid proteins containing ColQ peptide insertions were also evaluated (LBV31).

[0193] Vector genome yields and peptides are summarized in Table 2. Table 2: Alternative RGD-presenting scaffolds [Table 2]

[0194] The results are shown in Figures 6A to 6D. Significant enhancement of C2C12 transduction was observed for the VHH RGD inserted capsid LBV55 compared to wild type AAV9 5 days after transduction (Figures 6A and 6B). Additionally, significant enhanced transduction was observed for the modified PO1 capsid LBV31 compared to wild type AAVPO1 4 days after transduction (Figure 6C). Enhanced transduction was observed for the Rh774 derived LBV30 compared to Rh74 6 days after transduction. Example 3 Ex vivo imaging of AAV9, LBV30 and LBV31

[0195] To evaluate the in vivo targeting of the modified AAV capsids, mice were intravenously injected with the reporter virus at a dose of 1E12vg / mouse. Four weeks after injection, ex vivo imaging of skeletal muscle, smooth muscle, cardiac muscle, CNS, liver and other internal organs was performed. The results are shown in Figures 7A-7C. Figure 7A shows representative images comparing the modified capsids to the LBV30 and LBV31 variants. LBV30 is an AAV Rh74 capsid that contains a modified VP1 capsid protein with a ColQ peptide insertion and a VP2 / VP3 capsid protein with an RGD peptide insertion. LBV31 is an AAV PO1 capsid that contains a modified VP1 capsid protein with a ColQ peptide insertion and a VP2 / VP3 capsid protein with an RGD peptide insertion. The data in Figure 7B show that LBV31 has limited transduction of off-target tissues, while the data in Figure 7C show that LBV31 transduces target muscle. These data show that LBV31 shows a favorable profile with reasonable skeletal, smooth and cardiac muscle transduction and little or no transduction of multiple non-muscle tissues (most importantly, no liver transduction). LBV30 shows increased transduction of some muscles compared to AAV9. However, there is also a significant amount of liver transduction. Example 4 Liver-detargeting mutations in modified AAV capsid proteins

[0196] LBV55 presented enhanced muscle transduction. To enhance liver detargeting, mutations F501I, G505R and Y706C were introduced into VP1 and VP2 / VP3 of LBV55 to generate LBV91. Furthermore, LBV92 was generated from LBV30 by incorporating mutations F501I, G505R and Y706C into VP1 and VP2 / VP3. Viral vectors containing the luciferase reporting vector pAAV-tCAG firefly luciferase-P2A-Clover3 WPRE were prepared (Table 3) and administered to mice (Table 4). Mouse IV injection of luciferase reporter virus, 1E12vg / mouse. Ex vivo imaging of skeletal muscle, smooth muscle, cardiac muscle, CNS, liver and other internal organs was performed 2-4 weeks after injection. Table 3: AAV vectors used in the study [Table 3-1] [Table 3-2] Table 4: Study criteria, including time, dose and n. [Table 4]

[0197] LBV91 showed nearly complete liver detargeting, but muscle transduction was also reduced compared to LBV55. LBV92 showed maintained or enhanced muscle transduction and liver detargeting (less than 1% of Rh74 or AAV9). Figure 9. Thus, LBV92 achieves 1) increased muscle transduction (quad, tibialis anterior (TA), and diaphragm); 2) more than two orders of magnitude reduction in liver transduction; and 3) no increase in transduction in other organs.

Claims

1. i. VP1 capsid protein containing the acetylcholinesterase collagen-forming tail (ColQ) peptide sequence, ii. VP2 / VP3 capsid proteins containing the RGD motif peptide sequence AAV capsid, including

2. The ColQ peptide has an amino acid sequence 【Chemistry 1】 The AAV capsid according to claim 1, comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% the same amino acid sequence.

3. The AAV capsid according to claim 1, wherein the RGD motif insertion includes RGDLGLS (SEQ ID NO: 303), RGDLSTP (SEQ ID NO: 304), SNSRGDYNSL (SEQ ID NO: 305), ENRRGDFNNT (SEQ ID NO: 306), SRGDYNSL (SEQ ID NO: 307), RGDYNSL (SEQ ID NO: 308), RGDLST (SEQ ID NO: 309), or RGDYVGL (SEQ ID NO: 310).

4. The AAV capsid according to claim 1, wherein the peptide insertion comprises one or more linker sequences at the N-terminus or C-terminus of the inserted peptide.

5. The AAV capsid according to claim 1, which is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV Po1, AAVPHP.B, AAVrh74, or AAVrh10 capsid.

6. The AAV capsid according to claim 5, which is AAV Po1 or AAV rh74 capsid.

7. i) At least one of Rh74 F503I, G507I, Y707C, and / or Y708C, and / or ii) AAV9 N498I The AAV capsid according to claim 1, further comprising a mutation including the above.

8. The VP1 capsid protein contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs. 36-42, 44, 45, 47, 50, 51, 72, 74, or 75, and / or The VP2 / VP3 capsid protein contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by any one of SEQ ID NOs. 85-91, 93, 94, 96, 99, 100, 121, 123, or 124. The AAV capsid according to claim 1.

9. The VP1 capsid protein is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 134-140, 142, 143, 145, 148, 149, 170, 172, or 173, and / or The VP2 / VP3 capsid protein is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by any one of SEQ ID NOs: 182-188, 190, 191, 193, 196, 197, 218, 220, or 221. The AAV capsid according to claim 1.

10. An AAV virus vector comprising the AAV capsid according to claim 1, comprising a recombinant AAV (rAAV) vector encoding a therapeutic transgene or a target nucleotide sequence (NOI).

11. The aforementioned capsid, i) A VP1 capsid protein having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No. 36, ii) VP2 / VP3 capsid proteins having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No.

85. The AAV capsid according to claim 1, comprising:

12. The aforementioned capsid, i) A VP1 capsid protein having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No. 37, ii) VP2 / VP3 capsid proteins having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No.

86. The AAV capsid according to claim 1, comprising:

13. A cell comprising the AAV capsid described in claim 1 or the AAV viral vector described in claim 10.

14. A pharmaceutical composition comprising the AAV virus vector according to claim 11 and at least one pharmaceutically acceptable excipient and / or additive.

15. A method for treating a muscle and / or neuromuscular disorder in a subject requiring treatment for the muscle and / or neuromuscular disorder, comprising the step of administering a therapeutically effective amount of the AAV virus vector according to claim 10 to the subject.

16. i. VP1 capsid protein containing the acetylcholinesterase collagen-forming tail (ColQ) peptide sequence, ii. VP2 / VP3 capsid proteins containing the RGD motif peptide sequence AAV virus vector containing an AAV capsid.

17. A method for treating muscle and / or neuromuscular disorders in subjects requiring treatment of muscle and / or neuromuscular disorders, comprising the step of administering a therapeutically effective dose of an AAV virus vector to the subject, wherein the AAV virus vector is i. VP1 capsid protein containing the acetylcholinesterase collagen-forming tail (ColQ) peptide sequence, ii. VP2 / VP3 capsid proteins containing the RGD motif peptide sequence AAV capsid containing method.

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