Engineered Muscle-Targeting Compositions

Abstract

Described herein are compositions that include muscle-specific targeting moieties and muscle-specific targeting motifs. Also described herein are uses of compositions that include muscle-specific targeting motifs and muscle-specific targeting moieties. In some embodiments, muscle-specific targeting moieties and compositions that include muscle-specific targeting moieties can be used to deliver cargo directly to muscle cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 230,046, filed August 5, 2021, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named BROD-5450WP_ST26.txt, created on August 3, 2022, and having a size of 1,028,649 bytes (1 MB on disk). The contents of the Sequence Listing are incorporated herein by reference in their entirety.

[0003] The subject matter disclosed herein relates generally to muscle-targeting compositions comprising recombinant adeno-associated virus (AAV) vectors, as well as systems, compositions, and uses thereof. [Background technology]

[0004] Recombinant AAV (rAAV) is the most commonly used delivery vehicle for gene therapy and gene editing. Nevertheless, rAAV containing natural capsid mutants has limited cell tropism. In fact, currently used rAAV mainly infects the liver after systemic delivery. Furthermore, the transduction efficiency of conventional rAAVs with natural capsid mutants in other cell types, tissues, and organs is limited. Therefore, AAV-mediated polynucleotide delivery for diseases affecting cells, tissues, and organs other than the liver (e.g., nervous system, skeletal muscle, and cardiac muscle) typically requires a large amount of virus (typically about 1 × 10 14rAAV requires the injection of 1000 mg / kg of rAAV, which often results in liver toxicity. Furthermore, with conventional rAAV, the large amounts required make it extremely challenging to produce sufficient quantities of therapeutic rAAV needed to administer to adult patients. Furthermore, due to differences in gene expression and physiology, mouse and primate models respond differently to viral capsids. The transduction efficiency of different viral particles varies between different species, and as a result, preclinical trials in mice often do not accurately reflect the results in primates, including humans. Therefore, there is a need for improved rAAVs for use in the treatment of various genetic diseases. Summary of the Invention [Means for solving the problem]

[0005] In some embodiments, the composition comprises a targeting moiety effective for targeting a muscle cell, wherein the targeting moiety comprises one or more n-mer motifs, and at least one n-mer motif of the one or more n-mer motifs is selected from the group consisting of X m RGDX n and X m and X n are each independently selected from any amino acid, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and m is 1-4; and optionally, a cargo, wherein the cargo is linked or otherwise attached to the targeting moiety.

[0006] In some example embodiments, at least one n-mer motif is as in any one of Table 4, Table 5, Table 6, Figure 13, Figure 14F, Figure 19B, Figure 27B, Figure 28B, Figure 28D, or any combination thereof.

[0007] In some exemplary embodiments, the targeting moiety comprises a polypeptide, a polynucleotide, a lipid, a polymer, a sugar, or a combination thereof.

[0008] In some exemplary embodiments, the targeting moiety comprises a viral protein.

[0009] In some exemplary embodiments, the viral protein is a capsid protein.

[0010] In some exemplary embodiments, the viral protein is an adeno-associated virus (AAV) protein.

[0011] In some exemplary embodiments, the n-mer motif is located between two amino acids of a viral protein such that the n-mer motif is on the exterior of the viral capsid.

[0012] In some exemplary embodiments, the n-mer motif is inserted between any two consecutive amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide, or at an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide.

[0013] In some exemplary embodiments, the n-mer motif is inserted between amino acids 588 and 589 in the AAV9 capsid polypeptide or at a similar position in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide.

[0014] In some exemplary embodiments, the composition is an engineered viral particle.

[0015] In some exemplary embodiments, the engineered viral particle is an engineered AAV viral particle.

[0016] In some exemplary embodiments, the AAV viral particle is an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 viral particle.

[0017] In some exemplary embodiments, the n-mer motif is 3 to 15 amino acids.

[0018] In some example embodiments, the optional cargo is capable of treating or preventing a muscle disease or disorder.

[0019] In some example embodiments, the muscle disease or disorder is (a) an autoimmune disease; (b) a cancer; (c) a muscular dystrophy; (d) a neuromuscular disease; (e) a sugar or glycogen storage disease; (f) a repeat expansion disease; (g) a dominant-negative disease; (h) a cardiomyopathy; (i) a viral disease; (j) a premature aging disease; or (k) any combination thereof.

[0020] In some example embodiments, the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a PPMO, one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein comprising a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other recombinant or therapeutic RNA and / or protein, or any combination thereof.

[0021] In some exemplary embodiments, the cargo is capable of inducing exon skipping in a gene.

[0022] In some example embodiments, the cargo is capable of inducing exon skipping in the dystrophin gene.

[0023] In some exemplary embodiments, the cargo is a mini- or micro-dystrophin gene.

[0024] In some exemplary embodiments, the mini- or micro-dystrophin gene comprises spectrin-like repeats 1, 2, 3, and 24, and optionally an nNOS domain.

[0025] In some example embodiments, the repeat expansion disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD).

[0026] In some example embodiments, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or FSHD.

[0027] In some embodiments, the myotonic dystrophy is type 1 or type 2.

[0028] In some example embodiments, the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, or Danon disease.

[0029] In some embodiments, the sugar or glycogen storage disease is MPS type III disease or Pompe disease.

[0030] In some embodiments, the MPS III disease is MPS IIIA, IIIB, IIIC, or IIID.

[0031] In some example embodiments, the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia.

[0032] In some exemplary embodiments, the composition has increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof.

[0033] 1. A vector system comprising: a vector, the vector comprising: one or more polynucleotides each encoding all or part of one or more targeting moieties effective to target a muscle cell, each targeting moiety comprising one or more n-mer motifs, wherein at least one n-mer motif of the one or more n-mer motifs is selected from the group consisting of X m RGDX n and X m and X n are each independently selected from any amino acid, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and m is 1-4, and at least one of the one or more polynucleotides encodes at least one n-mer motif; and optionally, a regulatory element operably linked to one or more of the polynucleotides.

[0034] In some example embodiments, at least one n-mer motif is as in any one of Table 4, Table 5, Table 6, Figure 13, Figure 14F, Figure 19B, Figure 27B, Figure 28B, Figure 28D, or any combination thereof.

[0035] In some embodiments, the vector system further comprises a cargo.

[0036] In some exemplary embodiments, the cargo is a cargo polynucleotide, optionally linked to one or more polynucleotides encoding a targeting moiety.

[0037] In some exemplary embodiments, the cargo polynucleotide is present on the same vector as the one or more polynucleotides encoding the targeting moiety or on a different vector.

[0038] In some example embodiments, the vector system is capable of producing viral particles that contain the cargo when present.

[0039] In some exemplary embodiments, the vector system is capable of producing a capsid polypeptide that includes one or more targeting moieties.

[0040] In some exemplary embodiments, the vector system is capable of producing AAV viral particles.

[0041] In some exemplary embodiments, the AAV viral particle is an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 viral particle.

[0042] In some exemplary embodiments, the capsid polypeptide is an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 capsid polypeptide.

[0043] In some exemplary embodiments of the vector system, one or more polynucleotides encoding one n-mer motif are inserted between two codons corresponding to two amino acids of a viral protein, such that the n-mer motif is on the outside of the viral capsid.

[0044] In some exemplary embodiments, one or more polynucleotides encoding one or more n-mer motifs are inserted between two codons corresponding to any two consecutive amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide, or at analogous positions in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide.

[0045] In some exemplary embodiments, one or more polynucleotides encoding one or more n-mer motifs are inserted between the codons corresponding to amino acids 588 and 589 in the AAV9 capsid polynucleotide, or at analogous positions in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, and AAV rh.10 capsid polypeptides.

[0046] In some exemplary embodiments, the vectors comprising one or more polynucleotides each encoding all or part of one or more targeting moieties do not comprise splice control elements.

[0047] In some exemplary embodiments, the vector system further comprises a viral rep protein encoding polynucleotide.

[0048] In some exemplary embodiments, the viral rep protein encoding polynucleotide is an AAV rep protein encoding polynucleotide.

[0049] In some exemplary embodiments, the viral rep protein encoding polynucleotide is on the same vector or a different vector as one or more polynucleotides encoding all or part of one or more targeting moieties, respectively.

[0050] In some exemplary embodiments, the viral rep protein is operably linked to a regulatory element.

[0051] Polynucleotides encoded and / or produced by vector systems such as those in any of the preceding paragraphs or elsewhere herein are described in some example embodiments herein.

[0052] In some exemplary embodiments, the polypeptide is a viral polypeptide.

[0053] In some exemplary embodiments, the viral polypeptide is an AAV polypeptide.

[0054] In some example embodiments, described herein are particles produced by a vector system and / or comprising a polypeptide as described in any of the preceding paragraphs or elsewhere herein.

[0055] In some exemplary embodiments, the particle is a viral particle.

[0056] In some exemplary embodiments, the viral particle is an adeno-associated viral (AAV) particle, a lentiviral particle, or a retroviral particle.

[0057] In some embodiments, the viral particles have muscle-specific tropism.

[0058] In some example embodiments, the cargo is capable of treating or preventing a muscle disease or disorder.

[0059] In some example embodiments, the muscle disease or disorder is (a) an autoimmune disease; (b) a cancer; (c) a muscular dystrophy; (d) a neuromuscular disease; (e) a sugar or glycogen storage disease; (f) a repeat expansion disease; (g) a dominant-negative disease; (h) a cardiomyopathy; (i) a viral disease; (j) a premature aging disease; or (k) any combination thereof.

[0060] In some example embodiments, the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a PPMO, one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein comprising a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other recombinant or therapeutic RNA and / or protein, or any combination thereof.

[0061] In some exemplary embodiments, the cargo is capable of inducing exon skipping in a gene.

[0062] In some example embodiments, the cargo is capable of inducing exon skipping in the dystrophin gene.

[0063] In some exemplary embodiments, the cargo is a mini- or micro-dystrophin gene.

[0064] In some exemplary embodiments, the mini- or micro-dystrophin gene comprises spectrin-like repeats 1, 2, 3, and 24, and optionally an nNOS domain.

[0065] In some example embodiments, the repeat expansion disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD).

[0066] In some example embodiments, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or FSHD.

[0067] In some embodiments, the myotonic dystrophy is type 1 or type 2.

[0068] In some example embodiments, the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, or Danon disease.

[0069] In some embodiments, the sugar or glycogen storage disease is MPS type III disease or Pompe disease.

[0070] In some embodiments, the MPS III disease is MPS IIIA, IIIB, IIIC, or IIID.

[0071] In some example embodiments, the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia.

[0072] In some exemplary embodiments, the polypeptide, the particle, or both, has increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof.

[0073] In some example embodiments, described herein are cells comprising: (a) a composition described in any of the preceding paragraphs or elsewhere herein; (b) a vector system described in any of the preceding paragraphs or elsewhere herein; (c) a polypeptide described in any of the preceding paragraphs or elsewhere herein; (d) a particle described in any of the preceding paragraphs or elsewhere herein; or (e) any combination thereof.

[0074] In some exemplary embodiments, the cell is a prokaryotic cell. In some exemplary embodiments, the cell is a eukaryotic cell.

[0075] In some example embodiments, described herein are pharmaceutical formulations comprising: (a) a composition described in any of the preceding paragraphs or elsewhere herein; (b) a vector system described in any of the preceding paragraphs or elsewhere herein; (c) a polypeptide described in any of the preceding paragraphs or elsewhere herein; (d) a particle described in any of the preceding paragraphs or elsewhere herein; (e) a cell described in any of the preceding paragraphs or elsewhere herein; or (f) any combination thereof; and a pharmaceutically acceptable carrier.

[0076] In some example embodiments, methods are described herein that include administering to a subject in need thereof: (a) a composition described in any of the preceding paragraphs or elsewhere herein; (b) a vector system described in any of the preceding paragraphs or elsewhere herein; (c) a polypeptide described in any of the preceding paragraphs or elsewhere herein; (d) a particle described in any of the preceding paragraphs or elsewhere herein; (e) a cell described in any of the preceding paragraphs or elsewhere herein; (f) a pharmaceutical formulation described in the preceding paragraphs or elsewhere herein; or (g) any combination thereof.

[0077] In some example embodiments, the subject has a muscle disease or disorder.

[0078] In some example embodiments, the muscle disease or disorder is (a) an autoimmune disease; (b) a cancer; (c) a muscular dystrophy; (d) a neuromuscular disease; (e) a sugar or glycogen storage disease; (f) a repeat expansion disease; (g) a dominant-negative disease; (h) a cardiomyopathy; (i) a viral disease; (j) a premature aging disease; or (k) any combination thereof.

[0079] In some example embodiments, the repeat expansion disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD).

[0080] In some example embodiments, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or FSHD.

[0081] In some embodiments, the myotonic dystrophy is type 1 or type 2.

[0082] In some example embodiments, the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, or Danon disease.

[0083] In some embodiments, the sugar or glycogen storage disease is MPS type III disease or Pompe disease.

[0084] In some embodiments, the MPS III disease is MPS IIIA, IIIB, IIIC, or IIID.

[0085] In some example embodiments, the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia.

[0086] In some exemplary embodiments, the composition has reduced or eliminated targeting or specificity for non-muscle cells, hi some exemplary embodiments, the non-muscle cells are hepatocytes.

[0087] These and other embodiments, objects, features, and advantages of the example embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the illustrated example embodiments.

[0088] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention may be utilized and the accompanying drawings of which: [Brief explanation of the drawings]

[0089] [Figure 1] The adeno-associated virus (AAV) transduction machinery leads to the production of mRNA from the transgene.

[0090] [Figure 2] mRNA-based selection of AAV variants can be more stringent than DNA-based selection. The viral library was expressed under the control of a CMV promoter.

[0091] [Figure 3A-3B]Correlation between viral library and vector genomic DNA (Fig. 3A) and mRNA (Fig. 3B) in the liver.

[0092] [Figures 4A-4F] Capsid variants present at the DNA level and expressed at the mRNA level specific in various tissues. In this experiment, the viral library was expressed under the control of the CMV promoter.

[0093] [Figures 5A-5C] Capsid mRNA expression in various tissues under the control of cell-type-specific promoters (shown on the x-axis). CMV was included as an exemplary constitutive promoter. CK8 is a muscle-specific promoter. MHCK7 is a muscle-specific promoter. hSyn is a nerve-specific promoter. Expression levels from cell-type-specific promoters were normalized based on the expression levels from the constitutive CMV promoter in each tissue.

[0094] [Figure 6] FIG. 1 is a schematic diagram showing an embodiment of a method for generating and selecting capsid variants for tissue-specific gene delivery between species.

[0095] [Figure 7] Schematic diagram showing an embodiment for generating an AAV capsid mutant library, in particular, the insertion of random n-mers (n=3-15 amino acids) into wild-type AAV, e.g., AAV9.

[0096] [Figure 8] Schematic diagram showing an embodiment for generating an AAV capsid mutant library, in particular mutant AAV particle generation, where each capsid mutant encapsulates its own coding sequence as a vector genome.

[0097] [Figure 9]An exemplary vector map of a representative AAV capsid plasmid library vector (see, e.g., Figure 8) that can be used in an AAV vector system to generate an AAV capsid mutant library.

[0098] [Figure 10] Viral titers (calculated as AAV9 vector genomes / 15 cm dish) produced by constructs containing different constitutive and cell type-specific mammalian promoters.

[0099] [Figure 11] Schematic representation of the selection of further optimized myoAAV capsid mutants (also referred to herein as enhanced MyoAAV capsid mutants).

[0100] [Figure 12] Enhanced MyoAAV (eMyoAAV) capsid variants can transduce mouse muscle more efficiently compared to first-generation MyoAAV after systemic delivery.

[0101] [Figure 13] First and second generation myoAAV capsid mutants rely on the aVb6 integrin heterodimer for transduction of human primary myotubes (SEQ ID NOs: 2-12).

[0102] [Figures 14A-14F]DELIVER identifies a class of myotropic AAV capsid mutants containing an RGD motif. Figure 14A) Schematic of viral library generation and capsid mutant selection using DELIVER (SEQ ID NO: 13). Figure 14B) Comparison of rAAV titers generated using ITR-containing constructs expressing the AAV9 capsid coding sequence under the control of the CMV, CK8, or MHCK7 promoter. Data are presented as mean ± SD (n = 4). P values ​​are calculated by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (MCT). Figures 14C-14D) In ​​vivo expression of AAV9 capsid library mRNA expressed under the control of the CMV, CK8, or MHCK7 promoter in mouse skeletal muscle (Figure 14C) and heart (Figure 14D) after systemic injection. Data are presented as mean ± SD (n = 3). P values ​​are calculated by one-way ANOVA with Tukey's MCT. *: P<0.05, **: P<0.01. Figure 14E) Graph showing enrichment of capsid variants expressed under the MHCK7 promoter relative to the viral library at the DNA and mRNA levels in different mouse skeletal muscles. Figure 14F) Sequences of heptamer insertions in the top highly expressed capsid variants in mouse muscle after a second round of transcript-based selection. Mutants of the same color in each group are encoded by synonymous DNA codons (SEQ ID NOS: 8-12, 14-18). See also Figures 21A-21I.

[0103] [Figures 15A-15D]MyoAAV transduces mouse skeletal muscle with high efficiency after systemic injection. Figures 15A-15B) Whole-mount fluorescent images (Figure 15A) and cross-sections (Figure 15B) of skeletal muscle, heart, and liver from C57BL / 6J mice systemically injected with 1E+12vg of AAV9- or MyoAAV 1A-CMV-EGFP. Green: EGFP; red: laminin for muscle and lectin for liver; blue: Hoechst. Scale bar for cross-sections: 100 μm. Figure 15C) Quantification of the fold difference in EGFP mRNA expression in various tissues of injected male and female C57BL / 6J mice. The red dashed line indicates relative expression from AAV9-CMV-EGFP. Data are presented as mean ± SD (n = 3-4); *: P < 0.05, **: P < 0.01 (Student's t-test between mice injected with AAV9 and MyoAAV 1A for each group). Figure 15D) Quantification of in vitro transduction in mouse (left) and human (right) primary myotubes transduced with vehicle, AAV9, or MyoAAV 1A-CK8-Nluc. Data are presented as mean ± SD (n = 5); **: P < 0.01 (Student's t-test). Donor 1: 29-year-old male, Donor 2: 19-year-old female, Donor 3: 20-year-old male, Donor 4: 34-year-old female. See also Figures 22A-22K and 29A-29O.

[0104] [Figures 16A-16D]Systemic injection of MyoAAV results in rapid and sustained high-level reporter transgene expression in muscles throughout the body. Figure 16A) Whole-body in vivo bioluminescence images of BALB / cJ mice systemically injected with 4E+11vg of AAV8-, AAV9-, or MyoAAV 1A-CMV-Fluc, taken over 120 days. Figure 16B) Quantification of total luminescence from the forelimbs and hindlimbs of animals injected with AAV8-, AAV9-, or MyoAAV 1A-CMV-Fluc, assessed over 120 days. P values ​​were calculated between the AAV8, AAV9, and MyoAAV 1A groups by two-way ANOVA with Tukey's MCT; data are presented as mean ± SD (n = 5). **: P < 0.01 for both MyoAAV 1A vs. AAV8 and MyoAAV 1A vs. AAV9. The differences between the AAV8 and AAV9 groups were not statistically significant at any time point. Figure 16C) Whole-organ luminescence images of the TA, triceps, gastrocnemius, quadriceps, and abdominal muscles from mice injected with 4E+11vg of AAV8-, AAV9-, or MyoAAV-CMV-Fluc, harvested 4 months after injection. Color scale: 1E+7 to 1E+8. Figure 16D) Quantification of total luminescence from various muscles of animals injected with AAV8-, AAV9-, or MyoAAV-CMV-Fluc, harvested 120 days after injection. Data are expressed as mean ± SEM (n = 5). *: P<0.01 (Mann-Whitney test between MyoAAV and AAV9 groups). See also Figures 22A-22K, 23A-23B, and 29A-29O.

[0105] [Figures 17A-17O]Systemic administration of MyoAAV-Dmd CRISPR and MyoAAV-human MTM1 results in therapeutic effects in mouse models of DMD and XLMTM, respectively. Figure 17A) Representative immunofluorescence images of dystrophin (red) in mdx muscles injected with AAV9- or MyoAAV 1A-Dmd CRISPR. Scale bar: 400 μm. Figure 17B) Western blots detecting dystrophin and GAPDH in muscles from mice injected with AAV9- or MyoAAV 1A-Dmd CRISPR, with relative signal intensities determined by densitometry at the bottom. AU: arbitrary units, normalized to GAPDH. Figure 17C) Taqman-based quantification of exon 23 deletion mRNA in various muscles of adult mdx mice injected with AAV9- or MyoAAV 1A-Dmd CRISPR. Data are presented as mean ± SD (n = 9-10); **: P < 0.01 (Student's t test). Figures 17D-17E) Specific muscle strength of the tibialis anterior muscle (Figure 17D) and muscle force decline after six eccentric contractions (Figure 17E) for wild-type C57BL / 6J mice (n = 11) injected with vehicle, and mdx mice (n = 15) injected with vehicle, AAV9-Dmd CRISPR (n = 15), or MyoAAV 1A-Dmd CRISPR (n = 17). **: P < 0.01 (ANOVA with Tukey's MCT). F) Schematic of an experiment to examine the efficacy of 2E + 12 vg / kg of AAV9- or MyoAAV 1A-MHCK7-human MTM1 (hMTM1) systemically delivered to 4-week-old Mtm1 knockout (KO) mice. Figure 17G) Total body weight of Mtm1 KO mice injected with vehicle or 2E+12 vg / kg of AAV9- or MyoAAV 1A-MHCK7-hMTM1, and wild-type littermate controls injected with vehicle. Data are presented as mean ± SD (n = 6 for KO AAV9, n = 6 for KO MyoAAV 1A, n = 3 for wild-type vehicle, n = 3 for KO vehicle). P values ​​are calculated between MyoAAV 1A and AAV9 groups; **: P < 0.01 (multiple t-test using Holm-Sydak MCT).Figure 17H) Photographs of Mtm1 KO mice injected with either MyoAAV 1A-hMTM1 or AAV9-hMTM1 at 2E+12 vg / kg 16 weeks after virus injection. Figure 17K) Survival curves for Mtm1 KO animals injected with vehicle, AAV9-hMTM1, or MyoAAV-hMTM1, as well as vehicle-injected wild-type littermates. Data points for vehicle-injected Mtm1 KO mice are from a previous experiment. Figure 17I) Average hourly passive activity, assessed by running a running wheel in their cage, from vehicle-injected wild-type mice or Mtm1 KO mice injected with vehicle, AAV9-hMTM1, or MyoAAV 1A-hMTM1, both at 2E+12 vg / kg. Data are presented as mean ± SD for weekly measurements averaged over a 3-week period (n = 6 for KO AAV9, n = 6 for KO MyoAAV 1A, n = 3 for wild-type vehicle, n = 3 for KO vehicle). P values ​​are calculated between MyoAAV 1A and AAV9 groups; **: P < 0.01 (multiple t-test with Holm-Sydak MCT). Figure 17J) Number of rearing behaviors in a 5-minute period in an activity monitor by vehicle-injected wild-type mice or Mtm1 KO mice injected with AAV9-hMTM1 or MyoAAV-hMTM1, both at 2E+12 vg / kg. Data are presented as mean ± SEM for weekly measurements averaged over a 3-week period (n = 5 for KO AAV9, n = 6 for KO MyoAAV, n = 3 for wild-type vehicle). P values ​​are calculated between the MyoAAV and AAV9 groups; *: P<0.05, **: P<0.01 (two-stage Benjamini, Krieger, and Yekutieli test). Figure 17K) Survival curves for Mtm1 KO animals injected with vehicle, AAV9-hMTM1, or MyoAAV 1A-hMTM1, and wild-type littermates injected with vehicle. (n=6 for KO AAV9, n=6 for KO MyoAAV 1A, n=3 for wild-type vehicle, n=4 for KO vehicle). Data points for vehicle-injected Mtm1 KO mice are from a previous experiment.P values ​​are calculated between the MyoAAV 1A and AAV9 groups; **: P<0.01 (Mantel-Cox test). Figure 17L) Quantification of the fold difference in hMTM1 mRNA expression in the gastrocnemius, quadriceps, cardiac muscle, and liver of Mtm1 KO mice injected with AAV9- or MyoAAV 1A-hMTM1, analyzed 4 weeks after injection. The red dashed line indicates relative expression from AAV9-hMTM1. Data are expressed as mean ± SD (n=4); *: P<0.05, **: P<0.01 (Student's t-test between the AAV9- and MyoAAV 1A-injected groups for each tissue). Figure 17M) Quantification of vector genomes per diploid genome in various tissues of Mtm1 KO mice injected with AAV9- or MyoAAV 1A-hMTM1, analyzed 4 weeks after injection. Data are presented as mean ± SD (n = 4). *: P < 0.05, **: P < 0.01 (Student's t test). Figure 17N) Western blot detecting hMTM1 and GAPDH in muscles of Mtm1 KO mice injected with vehicle, AAV9-, or MyoAAV 1A-hMTM1, with relative signal intensities determined by densitometry at the bottom. AU: arbitrary units, normalized to GAPDH. Figure 17O) Specific muscle strength of the extensor digitorum longus (EDL) for wild-type C57BL / 6J mice injected with vehicle (n = 4) and Mtm1 KO mice injected with vehicle (n = 2), AAV9-hMTM1 (n = 4), or MyoAAV 1A-hMTM1 (n = 4). **: P < 0.01 (ANOVA with Tukey's MCT). See also Figures 24A-24G.

[0106] [Figures 18A-18L]MyoAAV transduction is dependent on both integrin heterodimers and AAVR. Figures 18A-18B) Quantification of in vitro transduction (Figure 18A) and cell surface-bound virus (Figure 18B) in HEK293 cells transfected with a plasmid encoding an RGD-binding integrin heterodimer or pUC19 and transduced with AAV9- or MyoAAV-CMV-Nluc. Data are presented as mean ± SEM (n = 3). P values ​​are calculated relative to pUC19-transfected cells in each group. *: P < 0.05, **: P < 0.01 (ANOVA with Dunnett's multiple comparison test). Figure 18C) In vitro transduction efficiency in mouse primary myotubes treated with various concentrations of the GLPG-0187 pan-integrin αV antagonist and transduced with AAV9- or MyoAAV-CK8-Nluc. Data are presented as mean ± SEM (n = 5). P values ​​are calculated relative to the 0 nM small molecule condition in each group. **: P < 0.01 (ANOVA with Dunnett's multiple comparison test). Figure 18D) In ​​vitro transduction efficiency in human primary myotubes treated with various concentrations of GLPG-0187 pan-integrin αV antagonist and transduced with AAV9- or MyoAAV 1A-CK8-Nluc. Data are presented as mean ± SD (n = 5). P values ​​are calculated relative to the 0 nM small molecule condition in each group. *: P < 0.01 (ANOVA with Dunnett's multiple comparison test). Figures 18E-18F) In vitro transduction efficiency in human primary myotubes transduced with MyoAAV 1A-CK8-Nluc (Figure 18E) or AAV9-CK8-Nluc (Figure 18F) incubated with various concentrations of αVb1, αVb3, αVb6, αVb8, or MBP recombinant proteins. Data are presented as mean ± SD (n = 5). *: P < 0.01, **: P < 0.001 (ANOVA using Dunnett's MCT with 0 nM recombinant protein set as control for each group). Figure 18G) In vitro transduction efficiency in human primary myotubes transduced with AAV9- or MyoAAV-CK8-Nluc treated with various concentrations of anti-αVb6 antibody. Data are presented as mean ± SEM (n = 5).P values ​​are calculated relative to the 0 ng / ul antibody condition in each group. **: P<0.001 (ANOVA with Dunnett's multiple comparison test). Figures 18G-18H) In vitro transduction efficiency in human primary myotubes treated with various concentrations of anti-αVb6 (Figure 18G) or isotype control (Figure 18H) antibodies and transduced with AAV9- or MyoAAV 1A-CK8-Nluc. Data are presented as mean ± SD (n=5). P values ​​are calculated relative to the 0 ng / ul antibody condition in each group; *: P<0.01, **: P<0.001 (ANOVA with Dunnett's multiple comparison test). Figures 18I-18J) Quantification of in vitro transduction (Figure 18I) and cell surface-bound virus (Figure 18J) in HEK293 cells transfected with a plasmid encoding an RGD-binding integrin heterodimer or pUC19 and transduced with MyoAAV 1A-CMV-Nluc. Data are presented as mean ± SD (n = 3); *: P < 0.01 (one-way ANOVA with Dunnett's MCT, with pUC19-transfected cells set as the control). Figure 18K) In vitro transduction efficiency in human primary myotubes treated with various concentrations of the CWHM-12 pan-integrin αV antagonist and transduced with AAV9 or MyoAAV 1A-CK8-Nluc. Data are presented as mean ± SD (n = 5). P values ​​are calculated relative to the 0 nM small molecule condition in each group. *: P<0.01 (ANOVA using Dunnett's MCT; Figure 18L) shows in vitro transduction efficiency in human primary myotubes treated with various concentrations of isotype control antibody and transduced with AAV9 or MyoAAV 1A-CK8-Nluc. Data are presented as mean ± SD (n=5). P values ​​are calculated relative to the 0 ng / ul antibody condition in each group; *: P<0.01, **: P<0.001 (ANOVA using Dunnett's MCT). See also Figures 25A-25K and 26A-26O.

[0107] [Figures 19A-19M]Further evolution of MyoAAV using DELIVER will generate more enhanced muscle-tropic capsid variants. Figure 19A) Structure of the AAV9 VR-VIII surface loop and predicted structure of the MyoAAV 1A VR-VIII surface loop with amino acid annotation. Figure 19B) Schematic of the viral library design and sequences of the top hits identified from mouse muscle after a second viral library injection at two different doses (SEQ ID NOS: 2-7, 19-21). Figure 19C) Various tissues from C57BL / 6J mice systemically injected with 2E+11vg of MyoAAV 1A-CMV-EGFP (left) or MyoAAV 2A-CMV-EGFP (right) illuminated with blue light. Figure 19D) Whole-mount fluorescence images of the gastrocnemius, triceps, TA, and quadriceps muscles of mice systemically injected with 2E+11vg of AAV9-, MyoAAV 1A-, or MyoAAV 2A-CMV-EGFP. Figure 19E) Quantification of the fold difference in EGFP mRNA expression in various tissues of C57BL / 6J mice systemically injected with 2E+11vg of MyoAAV 1A- or MyoAAV 2A-CMV-EGFP compared to mice injected with the same dose of AAV9-CMV-EGFP. The red dashed line indicates relative expression from AAV9-CMV-EGFP. Data are presented as mean ± SD (n = 11 for MyoAAV 1A and MyoAAV 2A, n = 8 for AAV9). P values ​​are calculated compared to the AAV9 group. *: P<0.05, **: P<0.01 (ANOVA with Dunnett's MCT). Figure 19F) Quantification of in vitro transduction in human primary myotubes transduced with AAV9-, MyoAAV 1A-, or MyoAAV 2A-CK8-Nluc. Data are presented as mean ± SD (n=5). *: P<0.01 (ANOVA with Tukey's MCT). Figure 19G) In vitro transduction efficiency in human primary myotubes treated with various concentrations of GLPG-0187 integrin αV antagonist and transduced with AAV9- or MyoAAV 2A-CK8-Nluc. Data are presented as mean ± SD (n=5). P values ​​are calculated relative to the 0 nM small molecule condition in each group. *: P<0.05, **: P<0.01 (ANOVA with Dunnett's MCT).Figures 19H-19I) In vitro transduction efficiency in human primary myotubes transduced with MyoAAV 2A-CK8-Nluc (Figure 19H) or AAV9-CK8-Nluc (Figure 19I) incubated with various concentrations of αVb1, αVb3, αVb6, αVb8, or MBP recombinant proteins. Data are presented as mean ± SD (n = 5). **: P < 0.01, ***: P < 0.001 (one-way ANOVA with Dunnett's MCT using 0 nM recombinant protein condition in each group as control). Figure 19J) Whole-body in vivo bioluminescence images of BALB / cJ mice systemically injected with 2E+11 vg of AAVrh74-, AAV9-, or MyoAAV 2A-CMV-Fluc taken over 21 days. Color scale: 6E+6 to 1E+9. Figure 19K) Quantification of total luminescence from the hind paws of animals injected with AAVrh74-, AAV9-, or MyoAAV 2A-CMV-Fluc, assessed over 21 days. P values ​​are calculated between the AAVrh74, AAV9, and MyoAAV 2A groups by two-way ANOVA with Tukey's MCT; **: P<0.01 for both MyoAAV 2A vs. AAVrh74 and MyoAAV 2A vs. AAV9. The differences between the AAVrh74 and AAV9 groups are not statistically significant at any time point. Figure 19L) Quantification of vector genomes per diploid genome in various tissues of C57BL / 6J mice injected with 2E+11vg of AAV9-, MyoAAV 1A-, or MyoAAV 2A-CMV-EGFP. Data are presented as mean ± SD (n = 11 for MyoAAV 1A and MyoAAV 2A, n = 8 for AAV9). P values ​​are calculated between the MyoAAV 2A and AAV9 groups; *: P < 0.05, **: P < 0.01 (Student's t-test). Figure 19M) In vitro transduction efficiency in human primary myotubes transduced with AAV9- or first- or second-generation RGD-containing capsid mutants encoding Nluc under the control of the CK8 promoter, treated with various concentrations of anti-αVb6 antibody (left) or 10 ng / ul of isotype control antibody (right). Data are presented as mean ± SD (n = 5). P values ​​for the anti-αVb6 antibody data are calculated between the first- and second-generation groups.*: P<0.05, **: P<0.01 (Two-way ANOVA with Tukey's MCT). P values ​​for isotype control data are calculated between the isotype control and 10 ng / ul anti-αVb6 antibody conditions for each group; **: P<0.01 (Student's t-test). See also Figures 27A-27B.

[0108] [Figures 20A-20F]Systemic injection of EMyoAAV-CK8-microdystrophin at a low dose of 2E+13 vg / kg results in widespread microdystrophin expression and effective restoration of muscle function in adult DBA / 2J-mdx mice. Figure 20A) Representative immunofluorescence images for microdystrophin-FLAG (red) in muscles of DBA / 2J-mdx mice injected with 2E+13 vg / kg of AAV9- or MyoAAV 2A-CK8-microdystrophin. Scale bar: 400 μm. Figure 20B) Western blot detecting microdystrophin-FLAG and GAPDH in muscles of mice injected with 2E+13 vg / kg of AAV9- or MyoAAV 2A-CK8-microdystrophin, with relative signal intensities determined by densitometry at the bottom. AU: arbitrary units, normalized to GAPDH. Figure 20C) Quantification of the fold difference in microdystrophin mRNA expression in various muscles and livers of DBA / 2J-mdx mice systemically injected with 2E+13 vg / kg MyoAAV 2A-CK8-microdystrophin compared to mice injected with the same dose of AAV9-CK8-microdystrophin. The red dashed line indicates relative expression from AAV9-CK8-microdystrophin. Data are presented as mean ± SD (n = 9-10); *: P < 0.05, **: P < 0.01 (Student's t-test). Figure 20D) Quantification of vector genomes per diploid genome in various muscles and livers of DBA / 2J-mdx mice injected with 2E+13 vg / kg AAV9- or MyoAAV 2A-CK8-microdystrophin. Data are presented as mean ± SD (n = 9-10). **: P<0.01 (Student's t-test). Figures 20E-20F) Specific muscle strength (Figure 20E) and muscle strength after eccentric contractions (Figure 20F) were decreased in DBA2 / J mice (n=10) injected with vehicle and DBA / 2J-mdx mice (n=10) injected with vehicle (n=10), AAV9-CK8-microdystrophin (n=10), or MyoAAV 2A-CK8-microdystrophin (n=10). *: P<0.05, **: P<0.01, ***: P<0.001 (ANOVA with Tukey's MCT).

[0109] [Figures 21A-21I] DELIVER allows for the selection of capsid variants capable of functional transduction in various tissues. Figure 21A) Schematic of the various steps in AAV transduction. Figures 21B-21G) Graphs showing enrichment of capsid variants expressed under the ubiquitous CMV promoter relative to the viral library at the DNA and mRNA levels in the brain (Figure 21B), kidney (Figure 21C), lung (Figure 21D), skeletal muscle (Figure 21E), heart (Figure 21F), and liver (Figure 21G) of injected mice. The transcript-based selection used in DELIVER allows for more stringent identification of functional capsid variants compared to DNA-based selection. Enrichment relative to the viral library is calculated by dividing the reads per million (RPM) for each variant identified in the tissue by the RPM of the same variant in the viral library. Figures 21H-21I) Correlation between the abundance of each capsid variant in the viral library and the abundance of vector genomic DNA (Figure 21H) or expressed mRNA (under the control of the CMV promoter, Figure 21I) from that variant identified in the livers of mice injected with the viral library. While the relative amount of vector genomic DNA from each variant correlates with the abundance of that variant in the viral library, there is little correlation between the level of capsid mRNA expression from each variant and the amount of that variant in the viral library, based on linear regression between the two sample types. RPM: reads per million.

[0110] [Figures 22A-22K]MyoAAV produces recombinant AAV at similar titers compared to AAV9 and transduces muscle stem cells more effectively than AAV8 and AAV9 after systemic delivery to adult mdx-Ai9 mice. Figures 22A-22B) Whole-mount fluorescent images (Figure 22A) and cross-sections (Figure 22B) of triceps, gastrocnemius, and abdominal muscles from C57BL / 6J mice systemically injected with 1E+12vg of AAV9- or MyoAAV 1A-CMV-EGFP. Green: EGFP, red: laminin, blue: Hoechst. Scale bar for cross-sections: 100 μm. Figure 22C) Whole-mount fluorescent images of lung, kidney, spleen, and brain from C57BL / 6J mice systemically injected with 1E+12vg of AAV9- or MyoAAV 1A-CMV-EGFP. Figure 22D) Quantification of vector genomes per diploid genome in various tissues of C57BL / 6J mice injected with 1E+12vg of AAV9 or MyoAAV 1A-CMV-EGFP. Data are presented as mean ± SD (n = 4); *: P < 0.05, **: P < 0.01 (Student's t-test). Figure 22E) Comparison of recombinant AAV titers produced by the top RGD-containing capsid mutants with wild-type AAV9. Data are presented as mean ± SD (n = 3). P values ​​were calculated by ANOVA with Dunnett's MCT using AAV9 as a control. Figure 22F) Schematic of satellite cell transduction analysis experiments. Delivery of Cre recombinase into cells containing the Ai9 locus removes the STOP cassette from the genome and results in expression of tdTomato. Figure 22G) Percentage of tdTomato+ transduced muscle stem cells isolated from 6-month-old mdx-Ai9 mice 2 weeks after systemic injection of 4E+11vg of AAV8-, AAV9-, or MyoAAV 1A-CMV-Cre. Data are presented as mean ± SD (n=4); **: P<0.01 (one-way ANOVA with Tukey's MCT). Figure 22H) Representative FACS plot from muscle stem cells isolated from injected mdx-Ai9 mice. Figure 22I) Representative immunofluorescence image of myotubes differentiated from FACS-sorted muscle stem cells isolated from mdx-Ai9 mice systemically injected with AAV8-, AAV9-, or MyoAAV 1A-CMV-Cre. Green, myosin heavy chain (MHC); red, tdTomato; blue, Hoechst.Scale bar: 400 μm. Figure 22J) Western blot detecting EGFP and vinculin in muscle of mice injected with vehicle or 1E+12vg of AAV9- or MyoAAV 1A-CMV-EGFP. Figure 22K) Gating strategy for isolating skeletal muscle precursors from mononuclear cells in muscle.

[0111] [Figures 23A-23B] Systemic administration of MyoAAV results in long-term, high-level transgene expression in BALB / cJ mouse muscle. Figure 23A) Whole-body in vivo bioluminescence images of BALB / cJ mice injected with 4E+11 vg of AAV8-, AAV9-, or MyoAAV-CMV-Fluc over a 120-day period. This image shows the luminescence signal from the same mice shown in Figure 16A in a different color scale (5E+6 to 5E+7) to allow for detection of the signal in the muscles of mice injected with AAV8- and AAV9-CMV-Fluc. Figure 23B) Whole-organ luminescence images of the TA, triceps, gastrocnemius, quadriceps, and abdominal muscles from mice injected with 4E+11 vg of AAV8-, AAV9-, or MyoAAV-CMV-Fluc taken 4 months after injection. This image shows the luminescence signal from the same mouse shown in Figure 16C in various color scales (5E+5 to 5E+7) to allow detection of the signal in the muscle of mice injected with AAV8- and AAV9-CMV-Fluc.

[0112] [Figures 24A-24G]Systemic administration of MyoAAV-Dmd CRISPR results in higher levels of SaCas9 and gRNA expression compared to AAV9-Dmd CRISPR in multiple muscles throughout the body. (Figure 24A) Schematic of the AAV constructs used to generate AAV9- and MyoAAV 1A-Dmd CRISPR viruses. (Figure 24B) Schematic of dystrophin restoration after AAV-Dmd CRISPR administration to mdx mice. Mice were injected with 4.5E+12vg of SaCas9 and 9E+12vg of gRNA AAV. Figures 24C-24D) Quantification of the fold difference in SaCas9 mRNA (Figure 24C) or gRNA (Figure 24D) expression in various muscles of 8-week-old mdx mice systemically injected with 4.5E+12vg of AAV9- or MyoAAV 1A-CMV-SaCas9 and 9E+12vg of AAV9- or MyoAAV 1A-gRNA. The red dashed line indicates the relative expression from AAV9-CMV-SaCas9 (Figure 24F) and AAV9-gRNA (Figure 24F). Data are presented as mean ± SD (n = 5-6); *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 (Student's t-test). Figure 24E) Schematic of the AAV constructs used to generate AAV9- and MyoAAV 1A-MHCK7 human MTM1 viruses. Figure 24F) Schematic of the AAV constructs used to generate AAV9- and MyoAAV 1A-MHCK7 human MTM1 viruses. Figure 24G) Number of rearing behaviors in a 5-minute period by wild-type mice injected with vehicle, or Mtm1 KO mice injected with vehicle, AAV9-hMTM1, or MyoAAV 1A-hMTM1, both at 2E+12 vg / kg. Data are presented as mean ± SD for weekly measurements averaged over a 3-week period (n=6 for KO AAV9, n=6 for KO MyoAAV 1A, n=3 for wild-type vehicle, n=4 for KO vehicle). Data points for vehicle-injected Mtm1 KO mice are from a previous experiment. P values ​​are calculated between the MyoAAV 1A and AAV9 groups; **: P<0.01 (multiple t-test using Holm-Sydak MCT).

[0113] [Figures 25A-25K] Flow cytometry and Western blot confirm overexpression of integrin α and β proteins after plasmid transfection in HEK293 cells. (Figures 25A-25G) Representative histograms from flow cytometry analysis of HEK293 cells transfected with pUC19 or plasmids expressing integrin αV (Figure 25A), β1 (Figure 25B), α5 (Figure 25C), β8 (Figure 25D), β5 (Figure 25E), β3 (Figure 25F), or β6 (Figure 25G) under the control of the EF1α promoter and stained with antibodies against the overexpressed proteins. Figures 25H-25I) Western blot showing overexpression of integrins α8 (Figure 25H), αIIb (Figure 25I), β3 (Figure 25J), and β8 (Figure 25K) in HEK293 cells transfected with the corresponding integrin plasmids (lanes 1 and 2) compared to cells transfected with pUC19 (lanes 3 and 4).

[0114] [Figures 26A-26O]Integrin αV antagonists inhibit MyoAAV transduction, but not AAV9 transduction, in primary mouse myotubes and primary human myotubes from different donors. Figures 26A-26B) In vitro transduction efficiency in mouse primary myotubes treated with various concentrations of the integrin αV antagonists CWHM-12 (Figure 26A) or GLPG-0187 (Figures 26B and 26J) and transduced with AAV9 or MyoAAV-CK8-Nluc (MyoAAV 1A-CK8-Nluc). Data are presented as mean ± SEM (n = 5). *: P < 0.05, **: P < 0.01 (ANOVA using Dunnett's MCT with 0 nM condition for each group as control). Figures 26C-26H) In vitro transduction efficiency in human primary myotubes derived from three different donors treated with various concentrations of CWHM-12 (Figures 26C, 26E, and 26G) or GLPG-0187 (Figures 26D, 26F, and 26H) integrin αV antagonists and transduced with AAV9 or MyoAAV 1A-CK8-Nluc. Data are presented as mean ± SD (n=5). *: P<0.05, **: P<0.01 (ANOVA using Dunnett's MCT with 0 nM condition for each group as control). Figure 26I) Quantification of in vitro transduction in HEK293FT cells, HEK293FT AAVR KO cells, and HEK293FT AAVR KO cells overexpressing AAVR transfected with a plasmid encoding an RGD-binding integrin heterodimer or pUC19 and transduced with AAV9- or MyoAAV 1A-CMV-Nluc. Data are presented as mean ± SD (n = 3). **: P < 0.0001 (Student's t-test using log-transformed data). Figures 26J-26K) Quantification of in vitro transduction (Figure 26J) and binding (Figure 26K) of untreated HEK293 cells or cells pretreated with NA transduced with AAV2-, AAV9-, or MyoAAV 1A-CMV-Nluc. Data are expressed as mean ± SD (n=5); *: P<0.01, **: P<0.0001 (Student's t-test using log-transformed data).Figure 26L) Quantification of in vitro transduction (K) and binding (L) of HEK293 cells pretreated with NA and ECL or NA alone, along with AAV2-, AAV9-, or MyoAAV 1A-CMV-Nluc. Data are presented as mean ± SD (n = 5); *: P < 0.01, **: P < 0.0001 (Student's t-test using log-transformed data). Figure 26M) Comparison of in vitro transduction between HEK293FT and HEK293FT AAVR KO cells transduced with AAV2-, AAV4-, AAV9-, or MyoAAV 1A-CMV-Nluc. Data are presented as mean ± SD (n = 3). **: P < 0.0001 (Student's t-test using log-transformed data). Figure 26N) Quantification of in vitro binding of HEK293 cells pretreated with NA and ECL or NA alone, along with AAV2-, AAV9-, or MyoAAV 1A-CMV-Nluc. Data are presented as mean ± SD (n = 5); *: P < 0.01, **: P < 0.0001 (Student's t-test using log-transformed data). Figure 26O) Comparison of in vitro transduction between HEK293FT and HEK293FT AAVR KO cells transduced with AAV2-, AAV4-, AAV9-, or MyoAAV 1A-CMV-Nluc. Data are presented as mean ± SD (n = 3). **: P < 0.0001 (Student's t-test using log-transformed data).

[0115] [Figures 27A-27B]Second-generation RGD-containing capsid mutants are less dependent on αVβ6 for transduction of human primary myotubes compared to first-generation mutants. Figure 27A) Quantification of vector genomes per diploid genome in various tissues of C57BL / 6J mice injected with 2E+11vg of AAV9-, MyoAAV-, or EMyoAAV-CMV-EGFP. Data are presented as mean ± SEM (n = 11 for MyoAAV and EMyoAAV, n = 8 for AAV9). *: P < 0.05 (Mann-Whitney test). Figure 27B) In vitro transduction efficiency in human primary myotubes treated with various concentrations of anti-αVβ6 antibodies and transduced with AAV9- or first- or second-generation RGD-containing capsid mutants encoding Nluc under the control of the CK8 promoter. Data are presented as mean ± SEM (n = 5). The graph on the left shows the transduction efficiency for each individual mutant. In the graph on the right, results from the first-generation mutants (RGDLTTP (SEQ ID NO: 12), RGDLSTP (SEQ ID NO: 8), RGDLNQY (SEQ ID NO: 9), RGDATEL (SEQ ID NO: 10), and RGDTMSK (SEQ ID NO: 11)) and the second-generation mutants (GPGRGDQTTL (SEQ ID NO: 2), AEGRGDQYTR (SEQ ID NO: 3), ATGRGDLGQA (SEQ ID NO: 4), AVARGDQGLI (SEQ ID NO: 5), NISRGDQGYQ (SEQ ID NO: 6), APARGDQGSQ (SEQ ID NO: 7)) are plotted as two groups. *: P<0.01 (two-step Benjamini, Krieger, and Yekutieli test between the first- and second-generation groups) (SEQ ID NOs: 2-12).

[0116] [Figures 28A-28K]MyoAAV class capsid variants evolved in NHPs transduce various muscles of cynomolgus monkeys with high efficiency. Figures 28A-28B) Schematic diagram of viral library design and sequences of top hits identified from NHP muscle after two rounds of in vivo selection in cynomolgus monkeys from capsid variants containing random 7-mer inserts (Figure 28A) (SEQ ID NOS: 13, 22-27) or from a single round of in vivo selection in cynomolgus monkeys (Figure 28B) (SEQ ID NOS: 19, 28-32) using the top 120,000 variants identified from a first round of RGD-anchored selection in mice. Figure 28C) Comparison of in vitro transduction among 11 myotropic capsid variants selected in mice in human primary myotubes derived from four different donors. Data are presented as individual data points with the mean; **: P<0.01 (one-way ANOVA with Dunnett's MCT using MyoAAV 1A as a control). Figure 28D) Schematic of the pool of barcoded human frataxin transgene and capsid variants used to characterize the top muscle-tropic variants (SEQ ID NOs: 28, 30, 32-33) in NHPs. Figure 28E) Fold change in mRNA expression in response to AAVrh74 in different skeletal muscles, heart, and liver of three cynomolgus monkeys. mRNA expression is quantified by deep sequencing of the barcodes associated with each capsid variant. Data are presented as mean ± SD (n=3). *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 (One-way ANOVA with Dunnett's MCT using AAVrh74 as control). Figure 28F) Comparison of titers of AAVrh74, MyoAAV 3A, MyoAAV 4A, MyoAAV 4C, and MyoAAV 4E, and AAV9 with six different transgenes. Data are presented as mean ± SD (n = 6). *: P < 0.05; ****: P < 0.0001 (one-way ANOVA with Dunnett's MCT using AAVrh74 as control).Figure 28G) In vitro transduction efficiency in human primary myotubes treated with various concentrations of GLPG-0187 integrin αV antagonist and transduced with AAV9-, MyoAAV 3A-, MyoAAV 4A-, MyoAAV 4C-, or MyoAAV 4E-CK8-Nluc. Data are presented as mean ± SD (n = 5). P values ​​are calculated using one-way ANOVA with Dunnett's MCT, with the 0 nM condition set as the control for each group. : P < 0.0001 for MyoAAV 3A, MyoAAV 4A, MyoAAV 4C, and MyoAAV 4E; : P < 0.0001 for MyoAAV 4A and MyoAAV 4E. Figures 28H-28I) In vitro transduction efficiency in human primary myotubes transduced with AAV9-CK8-Nluc (Figure 28H) or MyoAAV 3A-, MyoAAV 4A-, MyoAAV 4C-, or MyoAAV 4E-CK8-Nluc (Figure 28I) incubated with various concentrations of αVb1, αVb3, αVb6, αVb8, or MBP recombinant proteins. Data are presented as mean ± SD (n = 5). **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 (one-way ANOVA with Dunnett's MCT using 0 nM recombinant protein condition in each group as control). Figure 28J) In vitro transduction efficiency in human primary myotubes treated with various concentrations of anti-αVb6 antibody or mouse isotype control and transduced with AAV9-, MyoAAV 3A-, MyoAAV 4A-, MyoAAV 4C-, or MyoAAV 4E-CK8-Nluc. Data are presented as mean ± SD (n = 5). : P < 0.0001 for MyoAAV 3A, MyoAAV 4A, MyoAAV 4C, and MyoAAV 4E; : P < 0.0001 for MyoAAV 3A, MyoAAV 4A, and MyoAAV 4E (one-way ANOVA with Dunnett's MCT using the 0 nM condition for each group as a control).Figure 28K) Comparison of in vitro transduction between HEK293FT and HEK293FT AAVR KO cells transduced with AAV2-, AAV4-, AAV9-, MyoAAV 3A-, MyoAAV 4A-, MyoAAV 4C-, or MyoAAV 4E-CMV-Nluc. Data are presented as mean ± SD (n = 4). *P < 0.0001 (Student's t-test on log-transformed data).

[0117] [Figures 29A-29O]MyoAAV 1A effectively transduces different skeletal muscles after systemic administration in mice from DBA / 2J and BALB / cJ backgrounds and is highly potent in muscle transduction after intramuscular delivery. Figures 29A-29D) Various tissues of female DBA / 2J (Figure 29A), male DBA / 2J (Figure 29B), female BALB / cJ (Figure 29C), and male BALB / cJ (Figure 29D) mice systemically injected with 1E+12 vg of AAV9-CMV-EGFP (top) or MyoAAV 1A-CMV-EGFP (bottom), illuminated by blue light. Figure 29E) Quantification of the fold difference in EGFP mRNA expression in the triceps, gastrocnemius, heart, and liver of male and female DBA / 2J and BALB / cJ mice systemically injected with 1E+12 vg of AAV9- or MyoAAV 1A-CMV-EGFP. The gray dashed line indicates relative expression from AAV9-CMV-EGFP. Data are presented as mean ± SD (n = 4-5); **: P < 0.01, ***: P < 0.001 (Student's t-test between mice injected with AAV9 and MyoAAV 1A for each group). Figure 29F) Quantification of the fold difference in EGFP mRNA expression in the TA of C57BL / 6J mice injected intramuscularly with 2E + 10 vg of AAV9- or MyoAAV 1A-CMV-EGFP. The gray dashed line indicates relative expression from AAV9-CMV-EGFP. Data are presented as mean ± SD (n = 5); ****: P < 0.0001 (Student's t-test). Figure 29G) Western blot detecting EGFP and tubulin in the TA muscle of C57BL / 6J mice injected intramuscularly with vehicle or 2E+10vg of AAV9- or MyoAAV 1A-CMV-EGFP. Figure 29H) Immunofluorescence images of TA from C57BL / 6J mice injected intramuscularly with vehicle or 2E+10vg of AAV9- or MyoAAV 1A-CMV-EGFP. Grayscale: EGFP, red: laminin, blue: Hoechst. Scale bar for cross sections: 400 μm. Figures 29I-29J) Quantification of ALT (Figure 29I) and AST (Figure 29J) enzyme levels in the serum of C57BL / 6J mice before and 14 and 28 days after systemic injection of vehicle or 1E+12vg of AAV9- or MyoAAV 1A-CMV-EGFP.Data are presented as mean ± SD (n = 5). P values ​​were calculated by two-way ANOVA with Tukey's MCT. Significance threshold: P < 0.05. Differences between any two groups at each time point were not significant. Figures 29K-29L) Inhibition of AAV9- or MyoAAV 1A-CMV-Nluc transduction in HEK293 cells by serum from mice injected with AAV9-CMV-EGFP (Figure 29K) or MyoAAV 1A-CMV EGFP (Figure 29L); data are presented as mean ± SD (n = 5); ****: P < 0.0001 (two-way ANOVA with Sidac's MCT). Figure 29M) Whole-organ luminescence images of the TA, triceps, gastrocnemius, quadriceps, and abdominal muscles from mice injected with 4E+11 vg of AAV8-, AAV9-, or MyoAAV 1A-CMV-Fluc, harvested 4 months after injection. Grayscale: 1E+7 to 1E+8. Figure 29N) Quantification of total luminescence from various muscles of animals injected with AAV8-, AAV9-, or MyoAAV 1A-CMV-Fluc, harvested 120 days after injection. Data are presented as mean ± SD (n=5); **: P<0.01, ***: P<0.001 (one-way ANOVA with Tukey's MCT). Figure 29O) Whole-organ luminescence images of TA, triceps, gastrocnemius, quadriceps, and abdominal muscles from mice injected with 4E+11vg of AAV8-, AAV9-, or MyoAAV 1A-CMV-Fluc, taken 4 months after injection. This image shows the luminescence signal from the same mice shown in Figure 17C in grayscale (5E+5 to 5E+7) to allow detection of signal in muscles of mice injected with AAV8- and AAV9-CMV-Fluc.

[0118] [Figures 30A-30C]Characterization of muscle-tropic capsid mutants evolved in cynomolgus monkeys. Figures 30A-30B) Fold change in mRNA expression for AAVrh74 in different tissues of C57BL / 6J mice (Figure 30A) or cynomolgus monkeys (Figure 30B). mRNA expression was quantified by deep sequencing of barcodes associated with each capsid mutant. Data are presented as mean ± SD (n=5 for mice and n=3 for cynomolgus monkeys). *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 (one-way ANOVA with Dunnett's MCT using AAVrh74 as control). Figure 30C) Representative immunofluorescence images for (ITGB6) integrin beta-6 (grayscale) and isotype control (grayscale) in C57BL / 6J mouse triceps, cynomolgus monkey triceps, and human scalene anterior muscle. Scale bar: 400 μm. DETAILED DESCRIPTION OF THE INVENTION

[0119] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0120] General definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2004. nd edition(1989)(Sambrook,Fritsch,and Maniatis);Molecular Cloning:A Laboratory Manual,4 thedition(2012)(Green and Sambrook);Current Protocols in Molecular Biology(1987)(F.M.Ausubel et al.eds.);the series Methods in Enzymology(Academic Press,Inc.):PCR 2:A Practical Approach(1995)(M.J.MacPherson,B.D.Hames,and G.R.Taylor eds.):Antibodies,A Laboratory Manual(1988)(Harlow and Lane,eds.):Antibodies A Laboratory Manual,2 ndedition 2013(EAGreenfield ed.);Animal Cell Culture(1987)(RIFreshney,ed.);Benjamin Lewin,Genes IX,published by Jones and Bartlett,2008(ISBN 0763752223);Kendrew et al.(eds.),The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd.,1994(ISBN 0632021829); Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons(New York, NY1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd edition (2011).

[0121] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0122] The term "optional" or "optionally" means that the subsequently described event, circumstance, or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0123] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. It will be further understood that the endpoints of each of the ranges are significant in relation to the other endpoint, and independently of the other endpoint. It is understood that there are a number of values ​​disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it will be understood that the particular value forms a further embodiment. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0124] It should be understood that such range formats are used for convenience and brevity and, therefore, should be interpreted in a flexible manner to include not only the numerical values ​​explicitly stated as range limits, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly written. To illustrate, a numerical range of "about 0.1% to 5%" should be interpreted not only to include the explicitly stated values ​​of about 0.1% to about 5%, but also to include individual values ​​(e.g., about 1%, about 2%, about 3%, and about 4%) and subranges within the stated range (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, as well as other possible subranges). When a range is expressed, a further embodiment includes from one particular value and / or to the other particular value.

[0125] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range (to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise), and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, when a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure; for example, the phrase "from x to y" includes ranges from "x" to "y" as well as ranges from greater than "x" to less than "y." Ranges may also be expressed as upper limits, e.g., "about x, y, z, or less," and should be interpreted to include the specific ranges of "about x," "about y," and "about z," as well as the ranges "less than x," "less than y," and "less than z." Similarly, the phrase "about x, y, z, or more" should be interpreted to include the specific ranges of "about x," "about y," and "about z," as well as the ranges "greater than x," "greater than y," and "greater than z." Furthermore, the phrase "about 'x' to 'y'," where 'x' and 'y' are numerical values, includes "about 'x' to about 'y'."

[0126] As used herein, the term "about" or "approximately," when referring to a measurable value such as a parameter, amount, temporal duration, etc., is intended to encompass variations at and from the stated value, for example, variations of + / - 10% or less, + / - 5% or less, + / - 1% or less, and + / - 0.1% or less, to the extent that such variations are appropriate for functioning in the disclosed invention. It should be understood that the value to which the "about" or "approximately" modifier refers is itself specifically and preferably disclosed. As used herein, the terms "about," "approximately," "at or about," and "substantially" can mean that the relevant amount or value can be the exact value or a value that provides a result or effect equivalent to that recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, and, where appropriate, reflect tolerances, conversion factors, rounding, measurement error, and the like, as well as other factors known to those skilled in the art to produce equivalent results or effects. In some situations, a value that will produce equivalent results or effects cannot be reasonably determined. Generally, amounts, sizes, formulations, parameters, or other quantities or characteristics are "about," "approximately," or "at or about," whether or not expressly stated as such. When "about," "approximately," or "at or about" is used before a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless otherwise specified.

[0127] As used herein, a "biological sample" may include whole cells and / or viable cells and / or cell debris. A biological sample may include (or be derived from) a "body fluid." The present invention encompasses embodiments in which the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female semen, gastric acid, gastric juice, lymph, mucus (including nasal discharge and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof. Biological samples include cell cultures, body fluids, and cell cultures from body fluids. Bodily fluids are obtained from a mammalian organism, for example, by lancing or other collection or sampling procedure.

[0128] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, apes, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0129] Various embodiments are described herein below. It should be noted that specific embodiments are not intended as an exhaustive description of the broader embodiments described herein or as limitations on such aspects. An embodiment described in conjunction with a particular embodiment is not necessarily limited to that embodiment and may be practiced with any other embodiment. Throughout this specification, references to "one embodiment," "an embodiment," or "example embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," or "example embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, although they may. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein may include certain features but not other features included in other embodiments, combinations of features from different embodiments are contemplated to be within the scope of the invention. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0130] All publications, published patent documents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0131] Overview Embodiments disclosed herein provide muscle-specific targeting moieties that can be linked or otherwise attached to cargo. Embodiments disclosed herein provide polypeptides and particles that can incorporate one or more muscle-specific targeting moieties. The polypeptides and / or particles can be linked, attached, encapsulate, or otherwise incorporate cargo, thereby linking the cargo to the targeting moiety.

[0132] The embodiments disclosed herein provide muscle-specific targeting moieties that can contain one or more of the n-mer motifs described further herein. In some embodiments, the n-mer motif is an enhanced myoAAV motif. In some embodiments, the n-mer motif can confer muscle specificity to the targeting moiety.

[0133] In some embodiments, the n-mer motif does not contain RGD as the first three amino acids of the motif. In some embodiments, the n-mer motif is a second-generation RGD motif. In some embodiments, the n-mer motif containing the second-generation RGD motif has higher muscle specificity, targeting, and / or efficacy than the n-mer motif not containing the second-generation RGD motif.

[0134] Embodiments disclosed herein provide engineered adeno-associated virus (AAV) capsids that can be engineered to confer cell-specific and / or species-specific tropism to the engineered AAV particle.

[0135] Embodiments disclosed herein also provide methods for generating rAAVs with engineered capsids, which may include systematically directing the generation of a diverse library of variants with modified surface structures, e.g., variant capsid proteins. Embodiments of the methods for generating rAAVs with engineered capsids may also include stringent selection of capsid variants capable of targeting specific cell, tissue, and / or organ types. Embodiments of the methods for generating rAAVs with engineered capsids may include stringent selection of capsid variants capable of efficient and / or uniform transduction in at least two or more species.

[0136] Embodiments disclosed herein provide vectors and systems capable of generating the engineered AAVs described herein.

[0137] Embodiments disclosed herein provide cells that may be capable of producing the engineered AAV particles described herein. In some embodiments, the cells contain one or more vectors or systems described herein.

[0138] The embodiments disclosed herein provide engineered AAVs that can include the engineered capsids described herein. In some embodiments, the engineered AAVs can include a cargo polynucleotide that is delivered to a cell. In some embodiments, the cargo polynucleotide is a recombinant polynucleotide.

[0139] Embodiments disclosed herein provide formulations that may contain an engineered AAV vector or system thereof, an engineered AAV capsid, an engineered AAV particle comprising an engineered AAV capsid described herein, and / or an engineered cell described herein comprising an engineered AAV capsid and / or an engineered AAV vector or system thereof. In certain embodiments, the formulation may also include a pharmaceutically acceptable carrier. The formulations described herein can be delivered to a subject or cell in need thereof.

[0140]

[0013] Embodiments disclosed herein also provide kits that include one or more of the polypeptides, polynucleotides, vectors, engineered AAV capsids, engineered AAV particles, cells, or other components described herein, and combinations thereof, and one or more of the pharmaceutical formulations described herein. In embodiments, one or more of the polypeptides, polynucleotides, vectors, engineered AAV capsids, engineered AAV particles, cells, and combinations thereof described herein may be presented as a combination kit.

[0141] Embodiments disclosed herein provide methods of using engineered AAVs with cell-specific tropism described herein, for example, to deliver therapeutic polynucleotides to cells. In this manner, the engineered AAVs described herein can be used to treat and / or prevent disease in a subject in need thereof. Embodiments disclosed herein also provide methods of delivering engineered AAV capsids, engineered AAV viral particles, engineered AAV vectors, or systems thereof and / or formulations thereof to cells. Also provided herein are methods of treating a subject in need thereof by delivering engineered AAV particles, engineered AAV capsids, engineered AAV capsid vectors, or systems thereof, engineered cells, and / or formulations thereof to the subject.

[0142] Additional features and advantages of the engineered AAVs of the embodiments and methods of making and using the engineered AAVs are further described herein.

[0143] Muscle-specific targeting moieties and compositions thereof Described herein are targeting moieties that may be capable of specifically targeting, binding to, associating with, or otherwise interacting specifically with muscle cells. In certain embodiments, the targeting moiety may be or include an n-mer motif.

[0144] In one embodiment, the n-mer motif contains a second generation RGD motif. The term "second generation RGD motif" refers to an n-mer motif that includes the presence of the amino acid motif RGD, and X mRGDX n and X m and X n are each independently selected from any amino acid, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and m is 1 to 4. Exemplary n-mer motifs and methods for generating and identifying suitable n-mer motifs capable of targeting muscle are described in more detail elsewhere herein.

[0145] In some embodiments, the targeting moiety may comprise two or more n-mer motifs. In some embodiments, the targeting moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more n-mer motifs. In some embodiments, all n-motifs comprised in the targeting moiety may be the same. In some embodiments, two or more n-mer motifs are comprised, at least two of the n-mer motifs are different from each other. In some embodiments, two or more n-mer motifs are comprised, all of the n-mer motifs are different from each other. In some embodiments, each n-mer motif comprised in the targeting moiety may be any one of those depicted in Table 4, Table 5, Table 6, Figure 13, Figure 14F, Figure 19B, Figure 27B, Figure 28B, Figure 28D, or any of those depicted in the figures and examples described elsewhere herein. In some embodiments, n-mer motifs containing a second-generation RGD motif confer greater muscle specificity, targeting, and / or efficacy than n-mer motifs that do not contain a second-generation RGD motif. In certain embodiments, n-mer motifs containing a second generation RGD motif confer greater muscle specificity, targeting, and / or efficacy than n-mer motifs having RGD as the first three amino acids of the motif.

[0146] In some embodiments, the first 1, 2, 3, or 4 amino acids of an n-mer motif may replace 1, 2, 3, or 4 amino acids of the polypeptide into which it is inserted and preceding the insertion site. In some embodiments, the amino acids of the n-mer motif that replace one or more amino acids of the polypeptide into which the n-mer motif is inserted are located before or immediately before "RGD" in the n-mer motif. For example, in one or more of the 10-mer inserts shown in Tables 4-6, the first three amino acids shown may replace one to three amino acids in the polypeptide into which they are inserted. Using AAV as another non-limiting example, one or more of the n-mer motifs may be inserted, for example, between amino acids 588 and 589 of the AAV9 capsid prolyl peptide, and this insert may replace amino acids 586, 587, and 588, such that the amino acid immediately preceding the n-mer motif after insertion is residue 585. It will be understood that this principle can be applied to any other insertion situation and is not necessarily limited to insertions between residues 588 and 589 of the AAV9 capsid or the equivalent position in another AAV capsid. It will further be understood that in certain embodiments, no amino acids in the polypeptide into which the n-mer motif is inserted are replaced with the n-mer motif.

[0147] The muscle-specific targeting moiety may be linked to or otherwise associated with the cargo. In some embodiments, one or more muscle-specific targeting moieties described herein are directly linked to the cargo. In some embodiments, one or more muscle-specific targeting moieties described herein are indirectly linked to the cargo, such as via a linker molecule. In some embodiments, one or more muscle-specific targeting moieties described herein are linked to, bound to, encapsulated in, and / or associated with a polypeptide or other particle containing the cargo.

[0148] Exemplary particles include, but are not limited to, viral particles (e.g., viral capsids, including bacteriophage capsids), polysomes, liposomes, nanoparticles, microparticles, exosomes, micelles, and the like. As used herein, the term "nanoparticle" includes nanoscale deposits of uniform or heterogeneous material. Nanoparticles can be regular or irregular in shape and can be formed from multiple co-precipitated particles forming composite nanoscale particles. Nanoparticles can be approximately spherical in shape or can have a composite shape formed from multiple co-precipitated approximately spherical particles. Exemplary shapes of nanoparticles include, but are not limited to, spheres, rods, ellipsoids, cylinders, discs, and the like. In certain embodiments, the nanoparticles have an approximately spherical shape.

[0149] As used herein, the term "specific" when used in reference to a described interaction between two moieties refers to a non-covalent physical binding of a first and second moiety, where the binding between the first and second moiety is at least 2 times stronger, at least 5 times stronger, at least 10 times stronger, at least 50 times stronger, at least 100 times stronger, or stronger than the binding of either moiety to most or all other moieties present in the environment in which the binding occurs. Under the conditions used, e.g., physiological conditions such as those inside a cell or consistent with cell survival, the equilibrium dissociation constant, Kd, ​​is greater than or equal to 10 -3 M or less, 10 -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 The binding of two or more entities can be considered specific if the binding strength is less than or equal to M. In certain embodiments, specific binding can be achieved through multiple weaker interactions (e.g., multiple individual interactions, where each individual interaction is greater than or equal to 10 -3(Characterized by a Kd greater than M. In certain embodiments, specific binding, which may be referred to as "molecular recognition," is a saturable binding interaction between two entities that depends on the complementary orientation of functional groups on each entity. Examples of specific interactions include primer-polynucleotide interactions, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metal-chelate interactions, hybridization between complementary nucleic acids, and the like.

[0150] In certain embodiments, in addition to the n-mer motif, the targeting moiety may comprise a polypeptide, a polynucleotide, a lipid, a polymer, a sugar, or a combination thereof.

[0151] In some embodiments, the targeting moiety is incorporated into a viral protein, such as a capsid protein, including, but not limited to, lentivirus, adenovirus, AAV, bacteriophage, and retrovirus proteins. In some embodiments, the n-mer motif is located between two amino acids of the viral protein, such that the n-mer motif is on the exterior of the viral capsid (i.e., displayed on the surface of the viral capsid).

[0152] In some embodiments, compositions containing one or more muscle-specific targeting moieties described herein have increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof. As used herein, the terms "muscle-specific," "muscle cell specificity," "muscle cell potency," and the like refer to the increased specificity, selectivity, or potency of the muscle-specific targeting moieties of the invention and compositions incorporating the muscle-specific targeting moieties for muscle cells relative to non-muscle cells. In some embodiments, the cell specificity, selectivity, or potency, or a combination thereof, of / in a muscle-specific targeting moiety described herein or a composition incorporating a muscle-specific targeting moiety is at least 2 to at least 500 times more specific, selective, and / or potent in muscle cells relative to non-muscle cells. In some embodiments, the specificity, selectivity, or potency of / for a muscle-specific targeting moiety described herein is at least 2-fold to / or 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 1 3, 34, 35, 36, 37, 38, 39, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 3, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 times more specific or selective.

[0153] In some embodiments, compositions containing one or more muscle-specific targeting moieties described herein have reduced non-muscle cell potency, non-muscle cell specificity, reduced immunogenicity, or any combination thereof. In some embodiments, compositions containing one or more muscle-specific targeting moieties described herein have at least 2-fold to at least 500-fold less specificity, selectivity, and / or potency for / in non-muscle cells compared to muscle cells. In some embodiments, the specificity, selectivity, or potency of / in a muscle-specific targeting moiety described herein is at least 2-fold to / or 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 times less specific or selective.

[0154] The immunogenicity of compositions incorporating muscle-specific targeting moieties can be reduced, for example, by 1 to 100-fold or more. In certain embodiments, immunogenicity is reduced by 1 to 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100-fold or more reduction.

[0155] Cargo can include any molecule that can be linked to or bind to the muscle-specific targeting moiety described herein. Cargo can include, but is not limited to, nucleotides, oligonucleotides, polynucleotides, amino acids, peptides, polypeptides, riboproteins, lipids, sugars, pharmaceutically active agents (e.g., drugs, imaging agents, and other diagnostic agents), chemical compounds, and combinations thereof. In some embodiments, the cargo is DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit the translation or transcription of critical tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, anticonvulsants, anti-inflammatory agents, antihistamines, anti-infectives, radiosensitizers, chemotherapeutics, radioactive compounds, imaging agents, and combinations thereof.

[0156] In some embodiments, the cargo is capable of treating or preventing a muscular disease or disorder. In some embodiments, the muscular disease or disorder is (a) an autoimmune disease; (b) a cancer; (c) a muscular dystrophy; (d) a neuromuscular disease; (e) a sugar or glycogen storage disease; (f) a repeat expansion disease; (g) a dominant-negative disease; (h) a cardiomyopathy; (i) a viral disease; (j) a progeria disease; or (k) any combination thereof. In some embodiments, the repeat expansion disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or FSHD. In some embodiments, the myotonic dystrophy is type 1 or type 2. In some embodiments, the sugar or glycogen storage disease is MPS type III disease or Pompe disease. In some embodiments, the MPS type III disease is MPS type IIIA, IIIB, IIIC, or IIID. In some embodiments, the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia.

[0157] In certain embodiments, the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a PPMO, one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein comprising a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other recombinant or therapeutic RNA and / or protein, or any combination thereof.

[0158] In certain embodiments, the cargo is capable of inducing exon skipping in a gene.

[0159] In certain embodiments, the cargo is capable of inducing exon skipping in the dystrophin gene.

[0160] In some embodiments, the cargo is a mini- or micro-dystrophin gene. In some embodiments, the mini- or micro-dystrophin gene comprises spectrin-like repeats 1, 2, 3, and 24, or a combination thereof, and optionally an nNOS domain.

[0161] Engineered viral capsids and encoding polynucleotides Described herein are various embodiments of engineered viral capsids, e.g., adeno-associated viral (AAV) capsids, that can be engineered to confer cell-specific tropism, e.g., muscle-specific tropism, to the engineered viral particle. The engineered viral capsid can be a lentivirus, retrovirus, adenovirus, or AAV capsid. The engineered capsid can be included in an engineered viral particle (e.g., an engineered lentivirus, retrovirus, adenovirus, or AAV viral particle) and can confer cell-specific tropism, reduced immunogenicity, or both to the engineered viral particle. The engineered viral capsids described herein can comprise one or more engineered viral capsid proteins described herein. The engineered viral capsids described herein can comprise one or more engineered viral capsid proteins described herein that can contain a muscle-specific targeting moiety that contains or is composed of an n-mer motif described elsewhere herein.

[0162] The engineered viral capsid and / or capsid protein may be encoded by one or more engineered viral capsid polynucleotides. In certain embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, an engineered lentiviral capsid polynucleotide, an engineered retroviral capsid polynucleotide, or an engineered adenoviral capsid polynucleotide. In certain embodiments, the engineered viral capsid polynucleotide (e.g., an engineered AAV capsid polynucleotide, an engineered lentiviral capsid polynucleotide, an engineered retroviral capsid polynucleotide, or an engineered adenoviral capsid polynucleotide) may comprise a 3' polyadenylation signal. The polyadenylation signal may be an SV40 polyadenylation signal.

[0163] The engineered viral capsid may be a mutant of a wild-type viral capsid. For example, in some embodiments, the engineered AAV capsid may be a mutant of a wild-type AAV capsid. In some embodiments, the wild-type AAV capsid may be composed of VP1, VP2, VP3 capsid proteins, or a combination thereof. In other words, the engineered AAV capsid may contain one or more mutants of wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. In some embodiments, the serotype of the reference wild-type AAV capsid may be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the serotype of the wild-type AAV capsid may be AAV-9. The engineered AAV capsid may have a different tropism from that of the reference wild-type AAV capsid.

[0164] An engineered viral capsid can contain 1 to 60 engineered capsid proteins. In some embodiments, an engineered viral capsid can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, an engineered viral capsid can contain 0 to 59 wild-type viral capsid proteins. In certain embodiments, the engineered viral capsid may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type viral capsid proteins.

[0165] In some embodiments, an engineered AAV capsid may contain between 1 and 60 engineered capsid proteins. In some embodiments, an engineered AAV capsid may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, the engineered AAV capsid may contain 0 to 59 wild-type AAV capsid proteins. In some embodiments, the engineered AAV capsid may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type AAV capsid proteins.

[0166] In some embodiments, the engineered viral capsid protein may have an n-mer amino acid motif, where n may be at least 3 amino acids. In some embodiments, n may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, the engineered AAV capsid may have a hexameric or heptameric amino acid motif. In some embodiments, the n-mer amino acid motif may be inserted between two amino acids in a wild-type viral protein (VP) (or capsid protein). In some embodiments, the n-mer motif may be inserted between two amino acids in a variable amino acid region in the viral capsid protein.

[0167] In one embodiment, the n-mer motif can be inserted between two amino acids in a variable amino acid region of an AAV capsid protein. The core of each wild-type AAV viral protein contains eight β-barrel motifs (βB-βI) and an α-helix (αA) that are conserved in autonomous parvovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structural variable regions (VRs) are present in the surface loops that connect the β-strands, which cluster together to generate local variations on the capsid surface. AAV has 12 variable regions (also called hypervariable regions) (see, e.g., Weitzman and Linden. 2011. "Adeno-Associated Virus Biology." In Snyder, RO, Moullier, P. (eds.) Totowa, NJ: Humana Press). In some embodiments, one or more n-mer motifs may be inserted between two amino acids in one or more of the 12 variable regions in the wild-type AVV capsid protein. In some embodiments, one or more n-mer motifs may each be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X, VR-XI, VR-XII, or a combination thereof. In some embodiments, an n-mer may be inserted between two amino acids in VR-III of the capsid protein. In certain embodiments, the engineered capsid may have an n-mer inserted between any two consecutive amino acids between amino acids 262 and 269, between any two consecutive amino acids between amino acids 327 and 332, between any two consecutive amino acids between amino acids 382 and 386, between any two consecutive amino acids between amino acids 452 and 460, between any two consecutive amino acids between amino acids 488 and 505, between any two consecutive amino acids between amino acids 545 and 558, between any two consecutive amino acids between amino acids 581 and 593, or between any two consecutive amino acids between amino acids 704 and 714 of the AAV9 viral protein.In some embodiments, the engineered capsid may have an n-mer inserted between amino acids 588-589 of the AAV9 viral protein. In some embodiments, the engineered capsid may have a heptamer motif inserted between amino acids 588-589 of the AAV9 viral protein. SEQ ID NO: 1 is a reference AAV9 capsid sequence for at least the insertion sites described above. It will be understood that n-mers may be inserted at analogous positions in AAV viral proteins of other serotypes. In some embodiments, as described above, n-mers may be inserted between any two consecutive amino acids within the AAV viral protein, and in some embodiments, the insertion is made in the variable region.

[0168] In some embodiments, the first 1, 2, 3, or 4 amino acids of an n-mer motif may replace 1, 2, 3, or 4 amino acids of the polypeptide into which it is inserted and preceding the insertion site. In some embodiments, the amino acids of the n-mer motif that replace one or more amino acids of the polypeptide into which the n-mer motif is inserted are located before or immediately before "RGD" in the n-mer motif. For example, in one or more of the 10-mer inserts shown in Tables 4-6, the first three amino acids shown may replace one to three amino acids in the polypeptide into which they are inserted. Using AAV as another non-limiting example, one or more of the n-mer motifs may be inserted, for example, between amino acids 588 and 589 of the AAV9 capsid polypeptide, replacing amino acids 586, 587, and 588, such that the amino acid immediately preceding the inserted n-mer motif is residue 585. It will be understood that this principle can be applied to any other insertion situation and is not necessarily limited to insertions between residues 588 and 589 of the AAV9 capsid or the equivalent position in another AAV capsid. It will further be understood that in certain embodiments, no amino acids in the polypeptide into which the n-mer motif is inserted are replaced with the n-mer motif.

[0169] SEQ ID NO: 1 AAV9 capsid reference sequence. [ka]

[0170] In certain embodiments, the n-mer can be an amino acid or any amino acid motif shown in, or encoded by, a nucleic acid shown in Table 4, Table 5, Table 6, Figure 13, Figure 14F, Figure 19B, Figure 27B, Figure 28B, Figure 28D, and / or the Examples herein. In certain embodiments, insertion of an n-mer into an AAV or other viral capsid can result in a cell, tissue, organ, specific engineered AAV or other viral capsid, or other composition comprising an n-mer motif or capsid protein of the invention. In certain embodiments, engineered capsids or other compositions containing n-mer motifs have specificity for bone tissue and / or cells, lung tissue and / or cells, liver tissue and / or cells, bladder tissue and / or cells, kidney tissue and / or cells, heart tissue and / or cells, skeletal muscle tissue and / or cells, smooth muscle and / or cells, neuronal tissue and / or cells, intestinal tissue and / or cells, pancreatic tissue and / or cells, adrenal tissue and / or cells, brain tissue and / or cells, tendon tissue or cells, skin tissue and / or cells, spleen tissue and / or cells, eye tissue and / or cells, blood cells, synovial cells, immune cells (including specificity for particular types of immune cells), and combinations thereof. In certain embodiments, engineered capsids or other compositions containing n-mer motifs have specificity for muscle cells, including, but not limited to, skeletal muscle tissue and / or cells and smooth muscle tissue and / or cells.

[0171] In some embodiments, the AAV capsid or other viral capsid or composition may be muscle-specific. In some embodiments, the muscle-specificity of the engineered AAV or other viral capsid or other composition is conferred by a muscle-specific n-mer motif incorporated into the engineered AAV or other viral capsid or other composition described herein. Without intending to be bound by theory, it is believed that the n-mer motif confers a 3D structure to or within a domain or region of the engineered AAV capsid or other viral capsid or other composition such that interaction of a viral particle or other composition containing the engineered AAV capsid or other viral capsid or other composition described herein has an increased or improved interaction (e.g., increased affinity) with cell surface receptors and / or other molecules on the surface of muscle cells. In some embodiments, the cell surface receptor is an AAV receptor (AAVR). In some embodiments, the cell surface receptor is a muscle cell-specific AAV receptor. In some embodiments, the cell surface receptor or other molecule is a cell surface receptor or other molecule that is selectively expressed on the surface of muscle cells. In some embodiments, the cell surface receptor or molecule is an integrin or dimer thereof, hi some embodiments, the cell surface receptor or molecule is a Vb6 integrin heterodimer.

[0172] In some embodiments, the muscle-specific engineered viral particles described herein or other compositions containing the muscle-specific capsids, n-mer motifs, or muscle-specific targeting moieties described herein may have increased uptake, delivery rate, transduction rate, efficiency, quantity, or a combination thereof in muscle cells compared to other cell types and / or other viral particles (including, but not limited to, AAV) and other compositions that do not contain the muscle-specific n-mer motifs of the invention.

[0173] Also described herein are polynucleotides described herein that encode the engineered muscle-specific targeting moieties and other compositions described herein, including but not limited to, engineered AAV capsids.

[0174] In certain embodiments, the engineered polynucleotide may be included in a polynucleotide configured to be a viral genome donor in a viral vector system that may be used to generate the engineered viral particles described elsewhere herein.

[0175] In some embodiments, an engineered AAV capsid-encoding polynucleotide can be included in a polynucleotide configured to be an AAV genome donor in an AAV vector system that can be used to generate engineered AAV particles described elsewhere herein. In some embodiments, the engineered AAV capsid-encoding polynucleotide can be operably linked to a polyadenylation tail. In some embodiments, the polyadenylation tail can be an SV40 polyadenylation tail. In some embodiments, the AAV capsid-encoding polynucleotide can be operably linked to a promoter. In some embodiments, the promoter can be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific for muscle (e.g., cardiac, skeletal, and / or smooth muscle), nerve and indicator cells (e.g., astrocytes, glial cells, Schwann cells, etc.), fat, spleen, liver, kidney, immune cells, cerebrospinal fluid cells, synovial cells, skin cells, cartilage, tendon, connective tissue, bone, pancreas, adrenal gland, blood cells, bone marrow cells, placenta, endothelial cells, and combinations thereof. In some embodiments, the promoter can be a constitutive promoter. Suitable tissue-specific and constitutive promoters are described elsewhere herein, are generally known in the art, and are commercially available.

[0176] Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and its variants, and SPc5-12 synthetic promoter.

[0177] Suitable immune cell-specific promoters include, but are not limited to, the B29 promoter (B cells), the CD14 promoter (monocytic cells), the CD43 promoter (leukocytes and platelets), CD68 (macrophages), and the SV40 / CD43 promoter (leukocytes and platelets).

[0178] Suitable blood cell-specific promoters include, but are not limited to, the CD43 promoter (leukocytes and platelets), the CD45 promoter (hematopoietic cells), INF-β (hematopoietic cells), the WASP promoter (hematopoietic cells), the SV40 / CD43 promoter (leukocytes and platelets), and the SV40 / CD45 promoter (hematopoietic cells).

[0179] Suitable pancreatic-specific promoters include, but are not limited to, the elastase-1 promoter.

[0180] Suitable endothelial cell-specific promoters include, but are not limited to, the Fit-1 promoter and the ICAM-2 promoter.

[0181] Suitable neural tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astrocytes), the SYN1 promoter (neurons), and NSE / RU5' (mature neurons).

[0182] Suitable kidney-specific promoters include, but are not limited to, the NphsI promoter (podocyte).

[0183] Suitable bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts).

[0184] Suitable lung-specific promoters include, but are not limited to, the SP-B promoter (lung).

[0185] Suitable liver-specific promoters include, but are not limited to, the SV40 / Alb promoter.

[0186] Suitable cardiac-specific promoters include, but are not limited to, α-MHC.

[0187] Suitable constitutive promoters include, but are not limited to, CMV, RSV, SV40, EF1α, CAG, and β-actin.

[0188] AAV with reduced non-myocyte specificity In certain embodiments, the n-mer motifs described herein are inserted into an AAV protein (e.g., an AAV capsid protein) that has reduced specificity (or undetectable, unmeasurable, or clinically irrelevant interaction) for one or more non-muscle cell types. Exemplary non-muscle cell types include, but are not limited to, liver, kidney, lung, heart, spleen, central or peripheral nervous system cells, bone, immune, stomach, intestinal, eye, skin cells, etc. In certain embodiments, the non-muscle cell is a hepatocyte.

[0189] In some exemplary embodiments, the AAV capsid protein is an engineered AAV capsid protein that has reduced or eliminated uptake in non-muscle cells compared to the corresponding wild-type AAV capsid polypeptide.

[0190] In some embodiments, the non-muscle cells are hepatocytes.

[0191] In some exemplary embodiments, the wild-type capsid polypeptide is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 capsid polypeptide.

[0192] In some exemplary embodiments, the engineered AAV capsid protein contains one or more mutations that result in reduced or eliminated uptake in non-muscle cells.

[0193] In some exemplary embodiments, the one or more mutations are at (a) position 267, (b) position 269, (c) position 504, (d) position 505, (e) position 590, (f), or any combination thereof, in the AAV9 capsid protein (SEQ ID NO: 1), or at one or more corresponding positions in a non-AAV9 capsid polypeptide.

[0194] In some exemplary embodiments, the non-AAV9 capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide.

[0195] In some exemplary embodiments, the mutation at position 267 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is an A to G or X mutation, where X is any amino acid.

[0196] In some exemplary embodiments, the mutation at position 269 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is an S or X to T mutation, where X is any amino acid.

[0197] In some exemplary embodiments, the mutation at position 504 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a G or X to A mutation, where X is any amino acid.

[0198] In some exemplary embodiments, the mutation at position 505 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a P or X to A mutation, where X is any amino acid.

[0199] In some exemplary embodiments, the mutation at position 590 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a Q or X to A mutation, where X is any amino acid.

[0200] In some exemplary embodiments, the engineered AAV capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 267, 269, or both of the wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 267 is a G to A mutation and the mutation at position 269 is an S to T mutation.

[0201] In some exemplary embodiments, the engineered AAV capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 590 of the wild-type AAV9 capsid protein (SEQ ID NO: 1), and the mutation at position 509 is a Q to A mutation.

[0202] In some exemplary embodiments, the engineered AAV capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 504, 505, or both of the wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 504 is a G to A mutation and the mutation at position 505 is a P to A mutation.

[0203] In certain embodiments, the AAV capsid protein into which the n-mer motif can be inserted can be 80-100 (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, to / or 100) percent identical to SEQ ID NO:4 or SEQ ID NO:5 of International Patent Application Publication No. WO 2019 / 217911, which is incorporated by reference as if set forth in its entirety herein. These sequences are also incorporated herein as SEQ ID NOs:330 and 331, respectively. When considering variants of these AAV9 capsid proteins with reduced liver specificity, it will be understood that residues 267 and / or 269 thereof must contain the relevant mutation or equivalent. SEQ ID NO: 330 [ka] SEQ ID NO: 331 [ka]

[0204] In certain embodiments, the AAV capsid protein into which the n-mer motif can be inserted can be 80-100 (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, to / or 100) percent identical to any of those described in Adachi et al., (Nat.Comm. 2014.5:3075, DOI:10.1038 / ncomms4075), which has reduced specificity for non-CNS cells, particularly hepatocytes. Adachi et al., (Nat.Comm. 2014.5:3075, DOI:10.1038 / ncomms4075) is incorporated herein by reference as if set forth in its entirety.

[0205] In some embodiments, the modified AAV has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 2 In certain embodiments, the modified AAV may have a 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent or fold reduction. In certain embodiments, the modified AAV may also have no measurable or detectable uptake and / or expression in one or more non-muscle cells.

[0206] How to generate engineered AAV capsids Methods for generating engineered AAV capsids are also provided herein. The engineered AAV capsid mutants can be mutants of wild-type AAV capsids. Figures 6-8 illustrate various embodiments of methods for generating engineered AAV capsids with mutant motifs, as described herein. Generally, an AAV capsid library can be generated by expressing engineered capsid vectors containing the engineered AAV capsid polynucleotides described above in a suitable AAV-producing cell line. See, for example, Figure 8. It will be understood that Figure 8 illustrates a helper-dependent method of AAV particle production, but this can also be performed by a helper-free method. This can generate an AAV capsid library that can contain another desired cell-specific engineered AAV capsid mutant. As shown in Figure 6, the AAV capsid library can be administered to various non-human animals for a first round of mRNA-based selection. As shown in Figure 1, the transduction process by AAV and related vectors can result in the production of mRNA molecules that reflect the genome of the virus that transduced the cell. As shown at least in the Examples herein, in contrast to simply detecting the presence of viral particles in cells by measuring the presence of viral DNA, mRNA-based selection is based on the functional product produced and can therefore be more specific and effective for determining viral particles that are capable of functionally transducing cells.

[0207] After the first administration, one or more engineered AAV viral particles with the desired capsid variants can then be used to form a filtered AAV capsid library. Desirable AAV viral particles can be identified by measuring the mRNA expression of the capsid variants and determining which variants are highly expressed in the desired cell type compared to undesired cell types. Those highly expressed in the desired cell, tissue, and / or organ type are the desired AAV capsid variant particles. In one embodiment, the AAV capsid variant-encoding polynucleotide is under the control of a tissue-specific promoter that has selective activity in the desired cell, tissue, or organ.

[0208] The engineered AAV capsid mutant particles identified from the first round can then be administered to various non-human animals. In some embodiments, the animals used in the second round of selection and identification are not the same as those used in the first round of selection and identification. As in the first round, after administration, top-expressing mutants in desired cells, tissues, and / or organ types can be identified by measuring intracellular viral mRNA expression. The top mutants identified after the second round can then be optionally barcoded and optionally pooled. In some embodiments, particularly if the end use of the top mutants is in humans, the top mutants from the second round can then be administered to non-human primates to identify the top cell-specific mutants. Each round of administration can be systemic.

[0209] In certain embodiments, a method of generating AAV capsid variants may include the steps of: (a) expressing a vector system described herein containing an engineered AAV capsid polynucleotide in a cell to generate engineered AAV viral particle capsid variants; (b) collecting the engineered AAV viral particle capsid variants generated in step (a); (c) administering the engineered AAV viral particle capsid variants to one or more first subjects, wherein the engineered AAV viral particle capsid variants are generated by expressing an engineered AAV capsid variant vector or system thereof in the cell and collecting the engineered AAV viral particle capsid variants produced by the cell; and (d) identifying one or more engineered AAV capsid variants that are produced at significantly higher levels by one or more particular cells or particular cell types in the one or more first subjects. In this context, "significantly higher" may be greater than or equal to about 2×10 per 15 cm dish. 11 ~about 6×10 12 The term may refer to a titer which may range from one vector genome to another.

[0210] The method may further include (e) administering some or all of the engineered AAV viral particle capsid mutants identified in step (d) to one or more second subjects; and (f) identifying one or more engineered AAV viral particle capsid mutants that are produced at significantly higher levels in one or more specific cells or specific cell types in the one or more second subjects. The cells in step (a) may be prokaryotic or eukaryotic. In some embodiments, the administration in step (c), step (e), or both, is systemic. In some embodiments, the one or more first subjects, the one or more second subjects, or both, are non-human mammals. In some embodiments, the one or more first subjects, the one or more second subjects, or both, are each independently selected from the group consisting of a wild-type non-human mammal, a humanized non-human mammal, a disease-specific non-human mammal model, and a non-human primate.

[0211] In some embodiments, further optimization of the mutant motifs may be performed, hi some embodiments, first and / or second generation motifs, including capsid RGD-containing motifs, may be further used to optimize capsid mutants, for example, as shown in Figure 11 and further described in the Examples herein.

[0212] The polynucleotide and vector systems described herein can also be used to generate viral particles and other compositions that can be generated to contain cargo molecules that can be delivered to cells.

[0213] Manipulation vectors and vector systems Also provided herein are vectors and vector systems that can contain one or more of the engineered polynucleotides described herein that can encode one or more of the n-mer motifs of the invention, including, but not limited to, engineered viral polynucleotides (e.g., engineered AAV polynucleotides). In certain embodiments, polynucleotides that can encode n-mer motifs of the invention can be any of those described in Table 4, Table 5, Table 6, Figure 13, Figure 14F, Figure 19B, Figure 27B, Figure 28B, Figure 28D, and / or described elsewhere herein. In certain embodiments, polynucleotides can encode any n-mer motif described in Table 4, Table 5, Table 6, Figure 13, Figure 14F, Figure 19B, Figure 27B, Figure 28B, Figure 28D, and / or described elsewhere herein. As used in this context, an engineered viral capsid polynucleotide refers to any one or more of the polynucleotides described herein that are capable of encoding an engineered viral capsid described elsewhere herein and / or that are capable of encoding one or more engineered viral capsid proteins described elsewhere herein. Furthermore, when a vector comprises an engineered viral capsid polynucleotide described herein, the vector may also be referred to and considered to be an engineered vector or system thereof, although not specifically designated as such. In embodiments, a vector may contain one or more polynucleotides encoding one or more elements of an engineered viral capsid described herein. The vectors and systems thereof may be useful for generating bacteria, fungi, yeast, plant cells, animal cells, and transgenic animals capable of expressing one or more components of the engineered viral capsids, particles, or other compositions described herein. Vectors containing one or more of the polynucleotide sequences described herein are within the scope of the present disclosure. One or more of the polynucleotides that are part of the engineered viral capsids and systems thereof described herein may be included in a vector or vector system.

[0214] In some embodiments, the vector may comprise an engineered viral (e.g., AAV) capsid polynucleotide having a 3' polyadenylation signal. In some embodiments, the 3' polyadenylation signal is an SV40 polyadenylation signal. In some embodiments, the vector does not have a splice control element. In some embodiments, the vector comprises one or more minimal splice control elements. In some embodiments, the vector may further comprise a modified splice control element, wherein the modification inactivates the splice control element. In some embodiments, the modified splice control element is a polynucleotide sequence sufficient to induce splicing between a rep protein polynucleotide and an engineered viral (e.g., AAV) capsid protein variant polynucleotide. In some embodiments, the polynucleotide sequence may be sufficient to induce splicing and is a splice acceptor or splice donor. In some embodiments, the viral (e.g., AAV) capsid polynucleotide is an engineered viral (e.g., AAV) capsid polynucleotide described elsewhere herein. In certain embodiments, the vector does not include one or more minimal splice control elements, modified splice control elements, splice acceptors, and / or splice donors.

[0215] The vectors and / or vector systems can be used, for example, to express one or more engineered viral (e.g., AAV) capsids and / or other polynucleotides in a cell, such as a producer cell, to generate engineered viral (e.g., AAV) particles and / or other compositions (e.g., polypeptides, particles, etc.) containing engineered viral (e.g., AAV) capsids or other compositions containing the n-mer motifs of the invention described elsewhere herein. Other uses for the vectors and vector systems described herein are also within the scope of this disclosure. In general, throughout this specification, the term is a tool that allows or facilitates the transfer of an entity from one environment to another. In some contexts, as will be understood by those skilled in the art, a "vector" can be a term of art that refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. A vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to effect replication of the inserted segment. Generally, a vector is capable of replication when linked to appropriate control elements.

[0216] Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that contain one or more free ends or no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other species of polynucleotides known in the art. Another type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments have been inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

[0217] A recombinant expression vector can be comprised of a nucleic acid (e.g., a polynucleotide) of the invention in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector contains one or more regulatory elements operably linked to the nucleic acid sequence to be expressed, which can be selected based on the host cell to be used for expression. In recombinant expression vectors, "operably linked" and "operatively-linked" are used interchangeably herein and are further defined elsewhere herein. With respect to vectors, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to regulatory elements that allow for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or within a host cell when the vector is introduced into the host cell). Advantageous vectors include adeno-associated viruses, and such vector types can also be selected to target specific cell types, such as engineered viral (e.g., AAV) vectors containing engineered viral (e.g., AAV) capsid polynucleotides with desired cell-specific tropism. These and other embodiments of vectors and vector systems are described elsewhere herein.

[0218] In some embodiments, the vector may be a bicistronic vector. In some embodiments, a bicistronic vector may be used with one or more elements of the engineered viral (e.g., AAV) capsid system described herein. In some embodiments, expression of the elements of the engineered viral (e.g., AAV) capsid system described herein may be driven by a suitable constitutive or tissue-specific promoter. If the elements of the engineered viral (e.g., AAV) capsid system are RNA, their expression may be driven by a Pol III promoter, such as the U6 promoter. In some embodiments, the two are combined.

[0219] Cell-based vector amplification and expression Vectors can be designed for expression of one or more elements, such as the viral (e.g., AAV) capsid systems described herein or other compositions (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) comprising the n-mer motifs of the invention, in a suitable host cell. In certain embodiments, a suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. Vectors can be viral or non-viral based. In certain embodiments, a suitable host cell is a eukaryotic cell. In certain embodiments, a suitable host cell is a suitable bacterial cell. Suitable bacterial cells include, but are not limited to, bacterial cells from the Escherichia coli species of bacteria. Many suitable strains of E. coli are known in the art for expression of vectors. These include, but are not limited to, Pir1, Stbl2, Stbl3, Stbl4, TOP10, XL1 Blue, and XL10 Gold. In certain embodiments, a host cell is a suitable insect cell. Suitable insect cells include those from Spodoptera frugiperda. Suitable strains of S. frugiperda cells include, but are not limited to, Sf9 and Sf21. In some embodiments, the host cell is a suitable yeast cell. In some embodiments, the yeast cell may be derived from Saccharomyces cerevisiae. In some embodiments, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed for expressing vectors. Suitable mammalian cells include, but are not limited to, HEK293, Chinese hamster ovary cells (CHO), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEF).Suitable host cells are further described in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0220] In some embodiments, the vector may be a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987, EMBO J. 6:229-234), pMFa (Kuijan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp., San Diego, Calif.). As used herein, "yeast expression vector" refers to a nucleic acid containing one or more sequences encoding RNA and / or polypeptides, and may further contain any desired elements controlling expression of the nucleic acid, as well as any elements that allow replication and maintenance of the expression vector within yeast cells. Many suitable yeast expression vectors and their characteristics are known in the art; for example, various vectors and techniques are set forth in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, RG and Gleeson, MA (1991) Biotechnology (NY) 9(11):1067-72. Yeast vectors may contain, but are not limited to, a centromeric (CEN) sequence, an autonomously replicating sequence (ARS), a promoter, e.g., an RNA polymerase III promoter operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., an auxotrophic, antibiotic, or other selectable marker). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrating plasmids, yeast replicating plasmids, shuttle vectors, and episomal plasmids.

[0221] In some embodiments, the vector may be a baculovirus vector or expression vector suitable for expressing polynucleotides and / or proteins in insect cells. Baculovirus vectors available for expressing proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., 1983, Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39). rAAV (recombinant adeno-associated virus) vectors are preferably produced in insect cells, such as Spodoptera frugiperda Sf9 insect cells, and grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial suppliers, such as Sigma Aldrich (EX-CELL 405).

[0222] In some embodiments, the vector is a mammalian expression vector. In some embodiments, the mammalian expression vector is capable of expressing one or more polynucleotides and / or polypeptides in mammalian cells. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195). The mammalian expression vector may contain one or more suitable regulatory elements capable of controlling the expression of one or more polynucleotides and / or proteins in mammalian cells. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. Further details regarding suitable regulatory elements are provided elsewhere herein.

[0223] For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0224] In certain embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235-275), certain promoters for T-cell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Baneiji, et al., 1983, Cell 33:729-740; Queen and Baltimore, 1983, Cell 33:741-748), neural-specific promoters (e.g., neurofilament promoters; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreatic-specific promoters (Edlund, et al., 1989, Proc. Natl. Acad. Sci. USA 86:5473-5477), and the like. al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., whey promoters; U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters, such as the mouse hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-546), are also encompassed. With respect to these prokaryotic and eukaryotic vectors, reference is made to U.S. Pat. No. 6,750,059, the contents of which are incorporated herein by reference in their entirety. Other embodiments may employ viral vectors, and in this regard reference is made to U.S. Patent Application No. 13 / 092,085, the contents of which are incorporated herein by reference in their entirety.Tissue-specific regulatory elements are known in the art, and in this regard, reference is made to U.S. Patent No. 7,776,321, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, a regulatory element may be operably linked to one or more elements of an engineered AAV capsid system described herein so as to drive expression of one or more elements of the engineered AAV capsid system.

[0225] Vectors can be introduced and propagated in prokaryotes or prokaryotic cells. In some embodiments, prokaryotes are used to amplify copies of vectors that are introduced into eukaryotic cells or as intermediate vectors in the production of vectors that are introduced into eukaryotic cells (e.g., amplifying plasmids as part of a viral vector packaging system). In some embodiments, prokaryotes are used to amplify copies of vectors and express one or more nucleic acids, for example, to provide a source of one or more proteins for delivery to a host cell or host organism.

[0226] In certain embodiments, the vector may be a fusion vector or a fusion expression vector. In certain embodiments, a fusion vector adds several amino acids to a protein encoded by the vector, for example, to the amino terminus, carboxy terminus, or both of the recombinant protein. Such fusion vectors can serve one or more purposes, such as (i) increasing the expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and (iii) aiding in the purification of the recombinant protein by acting as a ligand in affinity purification. In certain embodiments, expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in Escherichia coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polynucleotides and / or proteins. In certain embodiments, a fusion expression vector can contain a proteolytic cleavage site, which can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein, allowing for separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion moiety following purification of the fusion polynucleotide or protein. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, to the target recombinant protein.Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0227] In certain embodiments, one or more vectors driving expression of one or more elements of an engineered viral (e.g., AAV) capsid system or other composition comprising an n-mer motif described herein are introduced into a host cell such that expression of the elements of the engineered delivery system described herein directs the formation of an engineered viral (e.g., AAV) capsid system or other composition comprising an n-mer motif described herein (including, but not limited to, engineered gene transfer agent particles described in more detail elsewhere herein). For example, different elements of an engineered viral (e.g., AAV) capsid system or other composition comprising an n-mer motif described herein can each be operably linked to separate regulatory elements in separate vectors. RNA of the different elements of the engineered delivery systems described herein can be delivered to an animal or mammal or cells thereof to generate an animal or mammal or cells thereof that incorporate one or more elements of the engineered viral (e.g., AAV) capsid system or other composition comprising an n-mer motif described herein, or that contain one or more cells that incorporate and / or express one or more elements of the engineered viral (e.g., AAV) capsid system or other composition comprising an n-mer motif described herein, constitutively, inducibly, or regulatably express the different elements of the engineered viral (e.g., AAV) capsid system or other composition comprising an n-mer motif described herein.

[0228] In certain embodiments, two or more elements expressed from the same or different regulatory elements may be combined in a single vector, along with one or more additional vectors providing any components of the system not included in the first vector. Engineered polynucleotides of the invention combined in a single vector may be arranged in any suitable orientation, e.g., one element may be located 5' to the second element ("upstream") or 3' to the second element ("downstream"). The coding sequence of one element may be located on the same or opposite strand as the coding sequence of the second element and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of transcripts encoding one or more engineered viral (e.g., AAV) capsid proteins or other compositions comprising n-mer motifs described herein embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In certain embodiments, engineered polynucleotides of the invention (including, but not limited to, engineered viral polynucleotides) may be operably linked to and expressed from the same promoter.

[0229] Vector characteristics A vector may include additional features that may confer one or more functionalities to the vector, the polynucleotide delivered, the viral particle produced therefrom, or the polypeptide expressed therefrom. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, etc. It will be understood by those skilled in the art that the design of an expression vector and the additional features included may depend on factors such as the choice of host cell to be transformed, the level of expression desired, etc.

[0230] Adjustment element In embodiments, the polynucleotides and / or vectors thereof described herein (including, but not limited to, engineered AAV capsid polynucleotides of the invention) may comprise one or more regulatory elements that may be operably linked to the polynucleotide. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, e.g., polyadenylation signals, and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest, such as muscle, nerve, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as cell cycle-dependent or developmental stage-dependent, which may or may not be tissue- or cell-type-specific. In certain embodiments, the vector includes one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Exemplary pol III promoters include, but are not limited to, U6 and H1 promoters.Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Enhancer elements, such as the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981), are also encompassed by the term "regulatory element."

[0231] In some embodiments, the regulatory sequences may be those described in U.S. Pat. No. 7,776,321, U.S. Patent Application Publication No. 2011 / 0027239, and PCT Publication No. WO 2011 / 028929, the contents of which are incorporated herein by reference in their entireties. In some embodiments, the vector may contain a minimal promoter. In some embodiments, the minimal promoter is a Mecp2 promoter, a tRNA promoter, or a U6 promoter. In further embodiments, the minimal promoter is tissue-specific. In some embodiments, the length of the vector polynucleotide, the minimal promoter, and the polynucleotide sequence is less than 4.4 Kb.

[0232] To express a polynucleotide, a vector may include one or more transcriptional and / or translational initiation regulatory sequences, such as a promoter that directs transcription of a gene and / or translation of an encoded protein in a cell. In certain embodiments, a constitutive promoter may be used. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to, SV40, CAG, CMV, EF-1α, β-actin, RSV, and PGK. Suitable constitutive promoters for bacterial, yeast, and fungal cells are generally known in the art, such as the T-7 promoter for bacterial expression and the alcohol dehydrogenase promoter for expression in yeast.

[0233] In certain embodiments, the regulatory element may be a regulatable promoter. A "regulatable promoter" refers to a promoter that directs gene expression in a temporally and / or spatially regulated manner, but not constitutively, and includes tissue-specific, tissue-preferred, and inducible promoters. In certain embodiments, a regulatable promoter is a tissue-specific promoter as described elsewhere herein. Regulatable promoters include regulatable promoters and inducible promoters. In certain embodiments, a regulatable promoter may be used to direct expression of a polynucleotide in a particular cell type, under particular environmental conditions, and / or during a particular developmental stage. Suitable tissue-specific promoters include, but are not limited to, liver-specific promoters (e.g., APOA2, SERPIN A1 (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e.g., INS, IRS2, Pdx1, Alx3, Ppy), cardiac-specific promoters (e.g., Myh6 (α MHC), MYL2 (MLC-2v), TNI3 (cTnl), NPPA (ANF), Slc8a1 (Ncx1)), central nervous system cell promoters (e.g., SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3)), and the like. β), skin cell-specific promoters (e.g., FLG, K14, TGM3), immune cell-specific promoters (e.g., ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell-specific promoters (e.g., Pbsn, Upk2, Sbp, Fer114), endothelial cell-specific promoters (e.g., ENG), pluripotent and embryonic germ layer cell-specific promoters (e.g., Oct4, NANOG, synthetic Oct4, T-Brachyury, NES, SOX17, FOXA2, MIR122), and muscle cell-specific promoters (e.g., desmin). Other tissue- and / or cell-specific promoters are described elsewhere herein, may be generally known in the art, and are within the scope of the present disclosure.

[0234] An inducible / regulatable promoter can be a positive inducible / regulatable promoter (e.g., a promoter that activates transcription of a polynucleotide upon appropriate interaction with an activated activator, or inducer (compound, environmental condition, or other stimulus)) or a negative / regulatable inducible promoter (e.g., a promoter that is repressed (e.g., bound by a repressor) until the repressor condition of the promoter is removed (e.g., an inducing agent binds to a repressor bound to the promoter, stimulating release of the promoter by the repressor or removal of a chemical repressor from the promoter environment). The inducer can be a compound, environmental condition, or other stimulus. Thus, an inducible / regulatable promoter can respond to any suitable stimulus, such as a chemical, biological, or other molecular agent, temperature, light, and / or pH. Suitable inducible / regulatable promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE / OlexA, GVG, and pOp / LhGR.

[0235] When expression in plant cells is desired, the components of the engineered AAV capsid system described herein are typically placed under the control of a plant promoter, i.e., a promoter that can operate in plant cells.The use of different types of promoters is also contemplated.In some embodiments, the inclusion of engineered virus (e.g., AAV) capsid system vectors in plants can be for the purpose of viral vector production.

[0236] A constitutive plant promoter is a promoter capable of expressing the open reading frame (ORF) it controls in all or nearly all plant tissues during all or nearly all developmental stages of the plant (referred to as "constitutive expression"). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. Different promoters can direct the expression of genes in various tissues or cell types, or at different developmental stages, or in response to different environmental conditions. In certain embodiments, one or more of the engineered AAV capsid system components are expressed under the control of a constitutive promoter; for example, the cauliflower mosaic virus 35S promoter may be used to target enhanced expression in specific plant tissues, such as vascular cells in leaves or roots, or specific cell types in seeds. Examples of specific promoters for use in the engineered AAV capsid systems and other compositions of the present invention are found in Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire et al., (1992) Plant Mol Biol 20:207-18; Kuster et al., (1995) Plant Mol Biol 29:759-72; and Capana et al., (1994) Plant Mol Biol 25:681-91.

[0237] Examples of promoters that are inducible and can enable gene editing or spatiotemporal control of gene expression can use a form of energy. The form of energy can include, but is not limited to, acoustic energy, electromagnetic radiation, chemical energy, and / or thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), or light-inducible systems (phytochrome, LOV domain, or cryptochrome), such as light-inducible transcription effectors (LITEs) that direct changes in transcription activity in a sequence-specific manner. Components of light-inducible systems can include one or more elements of the engineered AAV capsid system described herein or other compositions of the present invention, a light-responsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcription activation / repression domain. In certain embodiments, the vector may comprise one or more of the inducible DNA binding proteins set forth in PCT Publication No. WO 2014 / 018423 and U.S. Patent Application Publication Nos. 2015 / 0291966, 2017 / 0166903, and 2019 / 0203212, which describe, for example, embodiments of inducible DNA binding proteins and methods of use that may be adapted for use with the present invention.

[0238] In some embodiments, transient or inducible expression can be achieved, for example, by including a chemically regulated promoter, i.e., where application of an exogenous chemical induces gene expression. Regulation of gene expression can also be achieved by including a chemically repressible promoter, where application of a chemical represses gene expression. Chemically inducible promoters include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-11-27, WO 93 / 01294), which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Antibiotic-regulated promoters, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156), may also be used herein.

[0239] In certain embodiments, a vector or system thereof may comprise one or more elements capable of translocating and / or expressing an engineered polynucleotide of the invention (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) to / in a particular cellular component or organelle, including, but not limited to, the nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus, chloroplasts, mitochondria, vacuoles, lysosomes, cytoskeleton, cell membrane, cell wall, peroxisomes, centrioles, etc.

[0240] Selectable Markers and Tags One or more of the engineered polynucleotides of the invention (e.g., engineered viral (e.g., AAV) capsid polynucleotides) can be operably linked, fused, or otherwise modified to include a polynucleotide encoding or being a selectable marker or tag, which can be a polynucleotide or a polypeptide. In certain embodiments, a polypeptide encoding a polypeptide selectable marker can be incorporated into an engineered polynucleotide (e.g., engineered viral (e.g., AAV) capsid polynucleotide) of the invention such that the selectable marker polypeptide, when translated, is inserted between the N- and C-termini of the engineered polypeptide (e.g., engineered AAV capsid polypeptide), or between two amino acids at the N- and / or C-termini of the engineered polypeptide (e.g., engineered AAV capsid polypeptide). In certain embodiments, the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).

[0241] It will be understood that polynucleotides encoding such selectable markers or tags can be incorporated into polynucleotides encoding one or more components of the engineered AAV capsid systems described herein in an appropriate manner to allow for expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be readily understood by those of skill in the art in light of the present disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of the present disclosure.

[0242] Suitable selectable markers and tags include, but are not limited to, affinity tags, such as chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) tags; solubilization tags, such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags, such as those composed of polyanionic amino acids, e.g., FLAG-tags; epitope tags, such as V5-tags, Myc-tags, HA-tags, and N-tags. E-tags; protein tags that may allow for specific enzymatic modification (such as biotinylation with biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging); DNA and / or RNA segments containing restriction enzyme or other enzyme cleavage sites; toxic compounds, including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT)); Examples of suitable markers include DNA segments encoding products that confer resistance to a substance; DNA and / or RNA segments encoding products that are originally missing in the recipient cell (e.g., tRN genes, auxotrophic markers); DNA and / or RNA segments encoding products that can be easily identified (e.g., phenotypic markers such as β-galactosidase and GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP), luciferase, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., juxtaposition of two DNA sequences that were not previously juxtaposed), DNA sequences that have not been acted on or have been acted on by restriction endonucleases or other DNA-modifying enzymes, chemicals, etc.; epitope tags (e.g., GFP, FLAG- and His-tags), and DNA sequences that create molecular barcodes or unique molecular identifiers (UMIs), including DNA sequences required for specific modifications (e.g., methylation) that enable their identification. Other suitable markers will be understood by those of skill in the art.

[0243] Selectable markers and tags can be operably linked to one or more components of the engineered AAV capsid system or other compositions and / or systems described herein via a suitable linker, e.g., a glycine or glycine-serine linker of about GS or GG up to (GGGGG)3 (SEQ ID NO: 35) or (GGGGS)3 (SEQ ID NO: 34). Other suitable linkers are described elsewhere herein.

[0244] A vector or vector system may comprise one or more polynucleotides encoding one or more targeting moieties. In certain embodiments, targeting moieties encoding polynucleotides may be included in a vector or vector system, such as a viral vector system, such that they are expressed within and / or on the generated viral particle, such that the viral particle can be targeted to a specific cell, tissue, organ, etc. In certain embodiments, targeting moieties encoding polynucleotides may be included in a vector or vector system such that an engineered polynucleotide of the invention (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) and / or a product expressed therefrom comprises a targeting moiety and can be targeted to a specific cell, tissue, organ, etc. In certain embodiments, such as non-viral carriers, the targeting moiety may be attached to a carrier (e.g., a polymer, lipid, inorganic molecule, etc.), which may enable targeting of the carrier and any attached or associated engineered polynucleotide, engineered polypeptide, or other composition of the invention described herein to a specific cell, tissue, organ, etc. In certain embodiments, the specific cell is a muscle cell.

[0245] Cell-free vectors and polynucleotide expression In some embodiments, polynucleotides encoding n-mer motifs of the present invention can be expressed from vectors or suitable polynucleotides in cell-free in vitro systems. In some embodiments, polynucleotides encoding one or more features of an engineered AAV capsid system can be expressed from vectors or suitable polynucleotides in cell-free in vitro systems. In other words, polynucleotides can be transcribed and, optionally, translated in vitro. In vitro transcription / translation systems and suitable vectors are generally known in the art and commercially available. In general, in vitro transcription and in vitro translation systems reproduce the respective processes of RNA and protein synthesis outside of a cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include promoter regulatory sequences that can be recognized and acted upon by T7, SP6, T3, or an appropriate polymerase to transcribe the polynucleotide or vector.

[0246] In vitro translation can be independent (e.g., translation of purified polyribonucleotides) or coupled / coupled to transcription. In certain embodiments, cell-free (or in vitro) translation systems can include extracts from rabbit reticulocytes, wheat germ, and / or Escherichia coli (E. coli). The extracts can contain various macromolecular components necessary for translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNAs, synthetases, initiation, elongation, and termination factors, etc.). Other components can be included in or added during the translation reaction, including, but not limited to, amino acids, energy sources (ATP, GTP), energy regeneration systems (creatine phosphate and creatine phosphokinase (eukaryotic systems)) (phosphoenolpyruvate and pyruvate kinase for bacterial systems), and other cofactors (Mg2+, K+, etc.). As described above, in vitro translation can be based on RNA or DNA starting materials. Some translation systems can use an RNA template as the starting material (e.g., reticulocyte lysate and wheat germ extract). Some translation systems can use a DNA template as the starting material (e.g., E. coli-based systems). In these systems, transcription and translation are coupled; DNA is first transcribed into RNA, which is then translated. Suitable standard and coupled cell-free translation systems are generally known in the art and commercially available.

[0247] Codon optimization of vector polynucleotides As described elsewhere herein, polynucleotides encoding the n-multimer motifs of the present invention and / or other polynucleotides described herein may be codon-optimized. In certain embodiments, polynucleotides of the engineered AAV capsid systems described herein may be codon-optimized. In certain embodiments, one or more polynucleotides contained in the vectors described herein ("vector polynucleotides"), in addition to optionally codon-optimized polynucleotides encoding n-multimers, including, but not limited to, embodiments of the engineered AAV capsid systems described herein, may be codon-optimized. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon (e.g., about or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit particular biases for particular codons for particular amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, inter alia, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in several ways. See Nakamura, Y., et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000).Codon computer algorithms are also available that optimize a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA / RNA-targeted Cas protein correspond to the most frequently used codon for a particular amino acid. For codon usage in yeast, see the online yeast genome database available at http: / / www.yeastgenome.org / community / codon_usage.shtml, or see "Codon selection in yeast," Bennetzen and Hall, J. Biol. Chem. 1982 Mar. 25;257(6):3026-31. For codon usage in plants, including algae, see Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 Jan;92(1):1-11.; and Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan 25;17(2):477-98; or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton BR, J Mol Evol. 1998 Apr;46(4):449-59.

[0248] A vector polynucleotide can be codon-optimized for expression in a particular cell type, tissue type, organ type, and / or subject type. In certain embodiments, the codon-optimized sequence is a sequence optimized for expression in a eukaryote, e.g., a human (i.e., optimized for expression in a human or human cells), or for another eukaryote, e.g., another animal (e.g., a mammal or bird), as described elsewhere herein. Such codon-optimized sequences are within the skill of one of ordinary skill in the art in light of the description herein. In certain embodiments, the polynucleotide is codon-optimized for a particular cell type. Such cell types may include, but are not limited to, epithelial cells (including skin cells, cells lining the digestive tract, cells lining other hollow organs), neural cells (nerves, brain cells, spinal column cells, neural support cells (e.g., astrocytes, glial cells, Schwann cells, etc.), muscle cells (e.g., cardiac, smooth muscle cells, and skeletal muscle cells), connective tissue cells (adipose and other soft tissue fill cells, bone cells, tendon cells, chondrocytes), blood cells, stem cells and other progenitor cells, immune system cells, embryonic cells, and combinations thereof. Such codon-optimized sequences are within the skill of one of ordinary skill in the art in view of the description herein. In certain embodiments, the polynucleotide is In some embodiments, the polynucleotide is codon-optimized for a specific organ. Such organs include, but are not limited to, muscle tissue, connective tissue, connective tissue, nervous tissue, and epithelial tissue. The creation of such codon-optimized sequences is within the skill of one of ordinary skill in the art in light of the description herein. In some embodiments, the polynucleotide is codon-optimized for a specific organ. Such organs include, but are not limited to, muscle, skin, intestine, liver, spleen, brain, lung, stomach, heart, kidney, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. The creation of such codon-optimized sequences is within the skill of one of ordinary skill in the art in light of the description herein.

[0249] In certain embodiments, the vector polynucleotide is codon-optimized for expression in a particular cell, such as a prokaryotic cell or a eukaryotic cell, which may be of or derived from a particular organism, such as a plant or a mammal, including but not limited to a human or a non-human eukaryote or animal or mammal described herein, e.g., a mouse, rat, rabbit, dog, livestock, or a non-human mammal or primate.

[0250] Non-viral vectors and carriers In some embodiments, the vector is a non-viral vector or carrier. In some embodiments, non-viral vectors may have reduced toxicity and / or immunogenicity and / or increased biosafety advantages compared to viral vectors. The term "non-viral vector and carrier," as used herein in this context, refers to molecules and / or compositions that are not based on a virus or one or more components of a viral genome (excluding any nucleotides delivered and / or expressed by a non-viral vector) that may be capable of binding, incorporating, linking, and / or otherwise interacting with an engineered capsid polynucleotide described herein (e.g., an engineered AAV capsid polynucleotide) or other composition of the invention, and that may be capable of transporting the polynucleotide into a cell and / or expressing the polynucleotide. It will be understood that this does not exclude the inclusion of a delivered virus-based polynucleotide. For example, if the delivered gRNA is directed against a viral component that is originally inserted or otherwise linked to a non-viral vector or carrier, this would not make the vector a "viral vector." Non-viral vectors and carriers include naked polynucleotides, chemical-based carriers, polynucleotide (non-viral)-based vectors, and particle-based carriers. It will be understood that the term "vector" when used in reference to non-viral vectors and carriers refers to a polynucleotide vector, and that "carrier" when used in reference thereto refers to a non-nucleic acid or polynucleotide molecule or composition that is bound to or otherwise interacts with the polynucleotide to be delivered, e.g., an engineered AAV capsid polynucleotide of the invention.

[0251] Naked polynucleotides In certain embodiments, one or more engineered AAV capsid polynucleotides described elsewhere herein or other polynucleotides of the invention can be included in a naked polynucleotide. As used herein, the term "naked polynucleotide" refers to a polynucleotide that is not associated with another molecule (e.g., a protein, lipid, and / or other molecule) that may often help protect it from environmental factors and / or degradation. As used herein, associated with includes, but is not limited to, linked to, adhered to, adsorbed to, enclosed in, enclosed in or within, mixed with, and the like. Naked polynucleotides comprising one or more engineered AAV capsid polynucleotides described herein or other polynucleotides of the invention can be delivered directly to and optionally expressed in a host cell. Naked polynucleotides can have any suitable two-dimensional and three-dimensional structure. By way of non-limiting example, a naked polynucleotide can be a single-stranded molecule, a double-stranded molecule, a circular molecule (e.g., plasmids and artificial chromosomes), a molecule comprising a single-stranded portion and a double-stranded portion (e.g., ribozyme), etc. In certain embodiments, a naked polynucleotide contains only an engineered AAV capsid polynucleotide or other polynucleotide of the invention. In certain embodiments, a naked polynucleotide can contain other nucleic acids and / or polynucleotides in addition to an engineered AAV capsid polynucleotide or other polynucleotide of the invention described elsewhere herein. A naked polynucleotide can include one or more elements of a transposon system. Transposons and systems thereof are described in more detail elsewhere herein.

[0252] Non-viral polynucleotide vectors In some embodiments, one or more engineered AAV capsid polynucleotides or other polynucleotides of the present invention may be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include, but are not limited to, transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, AR (antibiotic resistance)-free plasmids and mini-plasmids, covalently closed circular vectors (e.g., minicircles, minivectors, miniknots), linear covalently closed vectors ("dumbbell-shaped"), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (microlinear vector) vectors, ministrings, miniintron plasmids, PSK systems (post-segregational killing systems), ORT (operator repressor titration) plasmids, and the like. See, e.g., Hardee et al. 2017. Genes. 8(2):65.

[0253] In some embodiments, the non-viral polynucleotide vector may have a regulated origin of replication. In some embodiments, the non-viral polynucleotide vector may be an ORT plasmid. In some embodiments, the non-viral polynucleotide vector may have minimal immunologically defined gene expression. In some embodiments, the non-viral polynucleotide vector may have one or more post-segregational killing system genes. In some embodiments, the non-viral polynucleotide vector is AR-free. In some embodiments, the non-viral polynucleotide vector is a minivector. In some embodiments, the non-viral polynucleotide vector comprises a nuclear localization signal. In some embodiments, the non-viral polynucleotide vector may comprise one or more CpG motifs. In some embodiments, the non-viral polynucleotide vector may comprise one or more scaffold / substrate binding regions (S / MARs). See, for example, Mirkovitch et al. 1984. Cell. 39:223-232 and Wong et al. 2015. Adv. Genet. 89:113-152, whose techniques and vectors may be adapted for use in the present invention. S / MARs are AT-rich sequences that play a role in the spatial organization of chromosomes by binding DNA loop bases to the nuclear matrix. S / MARs are often found near regulatory elements such as promoters, enhancers, and DNA replication origins. Inclusion of one or more S / MARs can promote once-per-cell-cycle replication, maintaining the non-viral polynucleotide vector as an episome in daughter cells. In embodiments, the S / MAR sequence is located downstream of an actively transcribed polynucleotide (e.g., one or more engineered AAV capsid polynucleotides or other polynucleotides or molecules of the present invention) contained in the non-viral polynucleotide vector. In one embodiment, the S / MAR can be an S / MAR from the β-interferon gene cluster.See, for example, Verghese et al. 2014. Nucleic Acid Res. 42:e53; Xu et al. 2016. Sci. China Life Sci. 59:1024-1033; Jin et al. 2016. 8:702-711; Koirala et al. 2014. Adv. Exp. Med. Biol. 801:703-709; and Nehlsen et al. 2006. Gene Ther. Mol. Biol. 10:233-244, whose techniques and vectors can be adapted for use in the present invention.

[0254] In some embodiments, the non-viral vector is a transposon vector or system thereof. As used herein, "transposon" (also called a transposable element) refers to a polynucleotide sequence that can move from one location in a genome to another. There are several classes of transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require transcription of the polynucleotide to be moved (or transposed) to transpose the polynucleotide into a new genome or polynucleotide. DNA transposons do not require reverse transcription of the polynucleotide to be moved (or transposed) to transpose the polynucleotide into a new genome or polynucleotide. In some embodiments, the non-viral polynucleotide vector can be a retrotransposon vector. In some embodiments, the retrotransposon vector contains long terminal repeats. In some embodiments, the retrotransposon vector does not contain long terminal repeats. In some embodiments, the non-viral polynucleotide vector can be a DNA transposon vector. The DNA transposon vector can contain a polynucleotide sequence encoding a transposase. In some embodiments, the transposon-based vector is configured as a non-autonomous transposon-based vector, meaning that transposition does not occur naturally by itself. In some of these embodiments, the transposon-based vector lacks one or more polynucleotide sequences encoding proteins required for transposition. In some embodiments, the non-autonomous transposon-based vector lacks one or more Ac elements.

[0255] In certain embodiments, a non-viral polynucleotide transposon vector system may include a first polynucleotide vector containing an engineered AAV capsid polynucleotide described herein or other polynucleotide or molecule of the invention, flanked at the 5' and 3' ends by transposon terminal inverted repeats (TIRs), and a second polynucleotide vector comprising a polynucleotide capable of encoding a transposase linked to a promoter to drive expression of the transposase. When both are expressed in the same cell, the transposase may be expressed from the second vector and transpose material between the TIRs (e.g., engineered AAV capsid polynucleotides or other polynucleotides or molecules of the invention) in the first vector, integrating it into one or more locations in the genome of the host cell. In certain embodiments, the transposon vector or system thereof may be configured as a gene trap. In certain embodiments, the TIRs may be configured flanked by a strong splice acceptor site, followed by a reporter and / or other gene (e.g., one or more engineered AAV capsid polynucleotides or other polynucleotides or molecules of the invention) and a strong polyA tail. When transposition occurs using this vector or the system, the transposon can be inserted into an intron of a gene, and the inserted reporter or other gene can induce a mis-splicing process that, as a result, inactivates the trapped gene.

[0256] Any suitable transposon system can be used. Suitable transposons and systems can include the Sleeping Beauty transposon system (Tc1 / mariner superfamily) (see, e.g., Ivics et al. 1997. Cell. 91(4):501-510), piggyBac (piggyBac superfamily) (see, e.g., Li et al. 2013 110(25):E2279-E2287 and Yusa et al. 2011. PNAS. 108(4):1531-1536), Tol2 (see superfamily hAT), Frog Prince (Tc1 / mariner superfamily) (see, e.g., Miskey et al. 2003 Nucleic Acid Res. 31(23):6873-6881), and variants thereof.

[0257] Chemical Carriers In certain embodiments, the engineered AAV capsid polynucleotides or other polynucleotides or other molecules of the invention described herein can be linked to a chemical carrier. Chemical carriers that may be suitable for delivery of polynucleotides can be broadly divided into the following classes: (i) inorganic particles, (ii) lipid-based, (iii) polymer-based, and (iv) peptide-based. They can be classified into those (1) capable of forming a condensation complex with a polynucleotide (such as an engineered AAV capsid polynucleotide of the present invention), (2) capable of targeting specific cells, (3) capable of increasing delivery of a polynucleotide or other molecule of the present invention (such as an engineered AAV capsid polynucleotide) to the nucleus or cytoplasm of a host cell, (4) capable of degrading from DNA / RNA in the cytoplasm of a host cell, and (5) capable of sustained or controlled release. It will be understood that any given chemical carrier may comprise features from more than one category. The term "particle," as used herein, refers to a particle of any suitable size for delivery of the compositions of the present invention described herein (including particles, polypeptides, polynucleotides, and other compositions described herein). Suitable sizes include macro-, micro-, and nano-sized particles.

[0258] In some embodiments, the non-viral carrier can be an inorganic particle. In some embodiments, the inorganic particle can be a nanoparticle. The inorganic particle can be configured and optimized with variable size, shape, and / or porosity. In some embodiments, the inorganic particle is optimized to escape the reticuloendothelial system. In some embodiments, the inorganic particle can be optimized to protect encapsulated molecules from degradation. Suitable inorganic particles that can be used as non-viral carriers in this regard can include, but are not limited to, calcium phosphate, silica, metals (e.g., gold, platinum, silver, palladium, rhodium, osmium, iridium, ruthenium, mercury, copper, rhenium, titanium, niobium, tantalum, and combinations thereof), magnetic compounds, particles, and materials (e.g., supermagnetic iron oxide and magnetite), quantum dots, fullerenes (e.g., carbon nanoparticles, nanotubes, nanostrings, etc.), and combinations thereof. Other suitable inorganic non-viral carriers are described elsewhere herein.

[0259] In some embodiments, the non-viral carrier can be lipid-based. Suitable lipid-based carriers are also described in more detail herein. In some embodiments, the lipid-based carrier comprises a cationic lipid or an amphipathic lipid capable of binding to or otherwise interacting with negative charges on the polynucleotide to be delivered (e.g., an engineered AAV capsid polynucleotide of the present invention). In some embodiments, the chemical non-viral carrier system can comprise a polynucleotide (e.g., an engineered AAV capsid polynucleotide of the present invention or other composition or molecule) and a lipid (e.g., a cationic lipid). These are also referred to in the art as lipoplexes. Other embodiments of lipoplexes are described elsewhere herein. In some embodiments, the non-viral lipid-based carrier can be a lipid nanoemulsion. A lipid nanoemulsion can be formed by the dispersion of an immiscible liquid in another stabilizing emulsifier and can have approximately 200 nm particles composed of lipid, water, and surfactant that can contain the polynucleotide to be delivered (e.g., an engineered AAV capsid polynucleotide of the present invention). In some embodiments, the lipid-based non-viral carrier can be a solid lipid particle or nanoparticle.

[0260] In some embodiments, the non-viral carrier may be peptide-based. In some embodiments, the peptide-based non-viral carrier may contain one or more cationic amino acids. In some embodiments, 35-40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of the amino acids are cationic. In some embodiments, the peptide carrier may be used with other types of carriers (e.g., polymer-based carriers and lipid-based carriers) to functionalize these carriers. In some embodiments, the functionalization is to target host cells. Suitable polymers that may be included in polymer-based non-viral carriers include, but are not limited to, polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA), poly(DL-lactide-co-glycoside) (PLGA), dendrimers (see, e.g., U.S. Patent Application Publication No. 2017 / 0079916, whose technology and compositions may be adapted for use with the engineered AAV capsid polynucleotides of the invention), polymethacrylates, and combinations thereof.

[0261] In some embodiments, the non-viral carrier may be configured to release an engineered delivery system polynucleotide associated with or bound to the non-viral carrier in response to an external stimulus, such as pH, temperature, osmolality, the concentration of a particular molecule or composition (e.g., calcium, NaCl, etc.), pressure, etc. In some embodiments, the non-viral carrier may be a particle configured to include one or more of the engineered AAV capsid polynucleotides described herein or other compositions of the invention and an environmental trigger response element, and optionally a trigger. In some embodiments, the particle may include a polymer selected from the group of polymethacrylate and polyacrylate. In some embodiments, the non-viral particle may include one or more embodiments of the composition microparticles described in U.S. Patent Application Publication Nos. 20150232883 and 20050123596, the technology and compositions of which may be adapted for use in the present invention.

[0262] In certain embodiments, the non-viral carrier can be a polymer-based carrier. In certain embodiments, the polymer is cationic or predominantly cationic so that it can interact with the negatively charged polynucleotide (such as the engineered AAV capsid polynucleotides of the present invention) to be delivered in a charge-dependent manner. Polymer-based systems are described in more detail elsewhere herein.

[0263] viral vectors In certain embodiments, the vector is a viral vector. The term "viral vector," as used herein in this context, refers to a polynucleotide-based vector containing one or more elements from or based on one or more elements of a virus capable of expressing and packaging a polynucleotide, e.g., an engineered AAV capsid polynucleotide, cargo, or other composition or molecule of the invention, into a viral particle and generating said viral particle when used alone or with one or more other viral vectors (such as in a viral vector system). Viral vectors and systems thereof can be used to produce viral particles for delivery and / or expression and / or generation of one or more compositions of the invention described herein (including, but not limited to, any viral particles and associated cargo). A viral vector can be part of a viral vector system comprising multiple vectors. In certain embodiments, systems incorporating multiple viral vectors can enhance the safety of these systems. Suitable viral vectors can include adenovirus-based vectors, adeno-associated vectors, helper-dependent adenovirus (HdAd) vectors, hybrid adenovirus vectors, and the like. Other embodiments of viral vectors and viral particles produced therefrom are described elsewhere herein. In certain embodiments, the viral vectors are configured to produce replication-incompetent viral particles for improved safety of these systems.

[0264] Adenoviral vectors, helper-dependent adenoviral vectors, and hybrid adenoviral vectors In certain embodiments, the vector may be an adenoviral vector. In certain embodiments, the adenoviral vector may contain elements such that the vector or viral particles produced using the system may be serotype 2, 5, or 9. In certain embodiments, the polynucleotide delivered via the adenoviral particle may be up to about 8 kb. Thus, in certain embodiments, the adenoviral vector may contain a DNA polynucleotide to be delivered that may range in size from about 0.001 kb to about 8 kb. Adenoviral vectors have been used successfully in several situations (see, e.g., Teramato et al. 2000. Lancet. 355:1911-1912; Lai et al. 2002. DNA Cell. Biol. 21:895-913; Flotte et al., 1996. Hum. Gene. Ther. 7:1145-1159; and Kay et al. 2000. Nat. Genet. 24:257-261). An engineered AAV capsid can be included in an adenoviral vector to generate adenoviral particles containing the engineered AAV capsid.

[0265] In some embodiments, the vector may be a helper-dependent adenoviral vector or system thereof. These are also referred to in the art as "gutless" or "gutted" vectors, which are a modified generation of adenoviral vectors (see, e.g., Thrasher et al. 2006. Nature. 443:E5-7). In embodiments of a helper-dependent adenoviral vector system, one vector (the helper) may contain all viral genes necessary for replication but a conditional genetic defect in the packaging domain. The second vector in the system may contain only the ends of the viral genome, one or more engineered AAV capsid polynucleotides, and the native packaging recognition signal, which may allow selective packaged release from the cell (see, e.g., Cideciyan et al. 2009. N Engl J Med. 361:725-727). Helper-dependent adenoviral vector systems have been successful for gene delivery in some situations (see, e.g., Simonelli et al. 2010. J Am Soc Gene Ther. 18:643-650; Cideciyan et al. 2009. N Engl J Med. 361:725-727; Crane et al. 2012. Gene Ther. 19(4):443-452; Alba et al. 2005. Gene Ther. 12:18-S27; Croyle et al. 2005. Gene Ther. 12:579-587; Amalfitano et al. 1998. J. Virol. 72:926-933; and Morral et al. 1999. PNAS. 96:12816-12821). The techniques and vectors described in these publications can be adapted for the inclusion and delivery of the engineered AAV capsid polynucleotides described herein. In certain embodiments, polynucleotides delivered via helper-dependent adenoviral vectors or viral particles generated from such systems can be up to about 38 kb.Thus, in certain embodiments, the adenoviral vector can comprise a DNA polynucleotide to be delivered, which can range in size from about 0.001 kb to about 37 kb (see, e.g., Rosewell et al. 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001).

[0266] In some embodiments, the vector is a hybrid adenoviral vector or system. Hybrid adenoviral vectors combine the high transduction efficiency of gene-deleted adenoviral vectors with the long-term genomic integration potential of adeno-associated, retroviral, lentiviral, and transposon-based gene transfer. In some embodiments, such hybrid vector systems can provide stable transduction and limited integration sites. See, e.g., Balague et al. 2000. Blood. 95:820-828; Morral et al. 1998. Hum. Gene Ther. 9:2709-2716; Kubo and Mitani 2003. J. Virol. 77(5):2964-2971; Zhang et al. 2013. PloS One. 8(10)e76771; and Cooney et al. 2015. Mol. Ther. 23(4):667-674. The techniques and vectors described in these documents can be modified and adapted for use in the engineered AAV capsid system of the present invention. In certain embodiments, hybrid adenoviral vectors can comprise one or more features of retroviruses and / or adeno-associated viruses. In certain embodiments, hybrid adenoviral vectors can comprise one or more features of spumaretroviruses or foamy viruses (FVs). See, e.g., Ehrhardt et al. 2007. Mol. Ther. 15:146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841. The techniques and vectors described in these documents can be modified and adapted for use in the engineered AAV capsid system of the present invention. Advantages of using one or more features from FVs in hybrid adenoviral vectors or systems can include the ability of viral particles produced therefrom to infect a broad range of cells, a large packaging capacity as other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See also, for example, Ehrhardt et al. 2007. Mol. Ther. 156:146-156 and Shuji et al. 2011. Mol. Ther. 19:76-82.The techniques and vectors described in these documents can be modified and adapted for use in the engineered AAV capsid system of the present invention.

[0267] Adeno-associated vector In one embodiment, the manipulating vector or system thereof may be an adeno-associated vector (AAV). See, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351 (1994). While some of their characteristics are similar to adenovirus vectors, AAVs possess some deficiencies in their replication and / or pathogenicity and may therefore be safer than adenovirus vectors. In some embodiments, AAVs can integrate into specific sites on chromosome 19 in human cells without observable side effects. In some embodiments, the capacity of the AAV vector, system thereof, and / or AAV particle may be up to approximately 4.7 kb. The AAV vector or system may comprise one or more engineered capsid polynucleotides described herein.

[0268] An AAV vector or system thereof may include one or more regulatory molecules. In certain embodiments, the regulatory molecules may be promoters, enhancers, repressors, etc., which are described in more detail elsewhere herein. In certain embodiments, an AAV vector or system thereof may include one or more polynucleotides capable of encoding one or more regulatory proteins. In certain embodiments, the one or more regulatory proteins may be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. In certain embodiments, the promoter may be a tissue-specific promoter as described above. In certain embodiments, the tissue-specific promoter may drive expression of the engineered capsid AAV capsid polynucleotides described herein.

[0269] An AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein. The engineered capsid proteins can be capable of assembling into the protein shell (engineered capsid) of an AAV viral particle. The engineered capsid can have cell-specific, tissue-specific, and / or organ-specific tropism.

[0270] In some embodiments, the AAV vector or system may include one or more adenoviral helper factors or polynucleotides capable of encoding one or more adenoviral helper factors. Such adenoviral helper factors may include, but are not limited to, E1A, E1B, E2A, E4ORF6, and VA RNA. In some embodiments, the producer host cell line expresses one or more of the adenoviral helper factors.

[0271] The AAV vector or system can be configured to produce AAV particles with a specific serotype. In some embodiments, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof. The AAV of the AAV can be selected based on the cells to be targeted; for example, AAV serotype 1, 2, 5, 9, or hybrid capsid AAV-1, AAV-2, AAV-5, AAV-9, or any combination thereof can be selected to target brain and / or neural cells; AAV-4 can be selected to target cardiac tissue; and AAV-8 can be selected for delivery to the liver. Thus, in certain embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and / or neural cells can be configured to produce AAV particles having serotypes 1, 2, 5, or hybrid capsids AAV-1, AAV-2, AAV-5, or any combination thereof. In certain embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to produce AAV particles having the AAV-4 serotype. In certain embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to produce AAV particles having the AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21:75-80.

[0272] It will be understood that while different serotypes may provide a certain level of cell, tissue, and / or organ specificity, each serotype remains multitrophic and may therefore result in tissue toxicity when used to target tissues in which the serotype is less efficient at transducing. Thus, in addition to achieving some tissue targeting capability by selecting an AAV of a particular serotype, it will be understood that the tropism of an AAV serotype may be modified by the engineered AAV capsids described herein. As described elsewhere herein, mutants of wild-type AAV of any serotype may be generated by the methods described herein and determined to have a particular cell-specific tropism, which may be the same as or different from that of a reference wild-type AAV serotype. In certain embodiments, the cell, tissue, and / or specificity of a wild-type serotype may be enhanced (e.g., made more selective or specific for a particular cell type against which the serotype was already biased). For example, wild-type AAV-9 is biased toward muscle and brain in humans (see, e.g., Srivastava. 2017. Curr. Opin. Virol. 21:75-80). By including an engineered AAV capsid and / or capsid protein mutant of wild-type AAV-9 described herein, for example, the brain bias can be reduced or eliminated, and / or muscle specificity can be increased such that brain specificity is reduced in comparison, thus enhancing specificity for muscle compared to wild-type AAV-9. As described above, the inclusion of an engineered capsid and / or capsid protein mutant of a wild-type AAV serotype can have a different tropism than the wild-type reference AAV serotype. For example, an engineered AAV capsid and / or capsid protein mutant of AAV-9 can have specificity for tissues other than muscle or brain in humans.

[0273] In one embodiment, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAV is an AAV containing a genome with elements from one serotype packaged into a capsid derived from at least one different serotype. For example, if rAAV2 / 5 is produced and the production method is based on the helper-free transient transfection method described above, the first and third plasmids (adenosyl helper plasmids) will be the same as those described for rAAV2 production. However, the second plasmid, pRepCap, will be different. In this plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2 / 5, where the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is believed that the cell or tissue tropism exhibited by this AAV2 / 5 hybrid virus should be the same as that of AAV5. It will be appreciated that wild-type hybrid AAV particles suffer from the same specificity problems as the non-hybrid wild-type serotypes described above.

[0274] The advantages achieved by the wild-type-based hybrid AAV system can be combined with the increased customizable cell specificity that can be achieved with engineered AAV capsids, which can be combined by generating hybrid AAVs that can contain the engineered AAV capsids described elsewhere herein. It will be understood that the hybrid AAV can contain an engineered AAV capsid containing a genome with elements from a serotype different from the reference wild-type serotype of which the engineered AAV capsid is a mutant. For example, a hybrid AAV can be generated that contains an engineered AAV capsid that is a mutant of the AAV-9 serotype used to package a genome containing components from the AAV-2 serotype (e.g., rep elements). As with the wild-type-based hybrid AAVs described above, the tropism of the resulting AAV particles will be that of the engineered AAV capsid.

[0275] A table of specific wild-type AAV serotypes for these cells can be found in Grimm, D. et al, J. Virol. 82:5887-5911 (2008), reproduced below as Table 6. Further tropism details can be found in Srivastava. 2017. Curr. Opin. Virol. 21:75-80, as described above. [Table 1]

[0276] In one embodiment, the AAV vector or system is AAV rh.74 or AAV rh.10.

[0277] In some embodiments, the AAV vector or system thereof is configured as a "gutless" vector, similar to those described in connection with retroviral vectors. In some embodiments, a "gutless" AAV vector or system thereof may have cis-acting viral DNA elements involved in genome amplification and packaging in association with a heterologous sequence of interest (e.g., an engineered AAV capsid polynucleotide).

[0278] Vector construction The vectors described herein can be constructed using any suitable process or technique. In certain embodiments, one or more suitable recombination and / or cloning methods or techniques can be used with the vectors described herein. Suitable recombination and / or cloning techniques and / or methods can include, but are not limited to, those described in U.S. Patent Application Publication No. 2004-0171156 A1. Other suitable methods and techniques are described elsewhere herein.

[0279] The construction of recombinant AAV vectors is described in several publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any of the techniques and / or methods can be used and / or adapted to construct AAV or other vectors described herein. AAV vectors are described elsewhere herein.

[0280] In certain embodiments, a vector can have one or more insertion sites, e.g., restriction endonuclease recognition sequences (also called "cloning sites"). In certain embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors.

[0281] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of the engineered AAV capsid system described herein are used in the above-referenced documents, such as WO 2014 / 093622 (PCT / US2013 / 074667), and are described in more detail herein.

[0282] Virus particle generation from viral vectors AAV particle generation There are two main approaches for producing AAV particles from AAV vectors and systems such as those described herein, depending on how adenoviral helper factors are provided (helper versus helper-free). In certain embodiments, methods for producing AAV particles from AAV vectors and systems may involve adenoviral infection in a cell line that stably harbors an AAV replication- and capsid-encoding polynucleotide along with an AAV vector containing a polynucleotide to be packaged and delivered by the resulting AAV particle (e.g., an engineered AAV capsid polynucleotide). In certain embodiments, methods for producing AAV particles from AAV vectors and systems may be "helper-free" methods, which involve co-transfection of three vectors (e.g., plasmid vectors): (1) an AAV vector containing a polynucleotide of interest (e.g., an engineered AAV capsid polynucleotide) between two ITRs; (2) a vector carrying an AAV Rep-Cap-encoding polynucleotide; and (3) a helper polynucleotide into a suitable production cell line. Those skilled in the art will appreciate the various methods and variations thereof, both helper and helper-free, and the distinct advantages of each system.

[0283] The engineered AAV vectors and systems described herein can be produced by any of these methods.

[0284] Vector and viral particle delivery The vectors described herein (including non-viral carriers) can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides encoded by the nucleic acids described herein (e.g., engineered AAV capsid system transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.), and viral particles (derived from viral vectors and systems, etc.).

[0285] One or more engineered AAV capsid polynucleotides can be delivered using adeno-associated virus (AAV), adenovirus, or other plasmid or viral vector types, as described above, particularly formulations and dosages from, for example, U.S. Patent Nos. 8,454,972 (formulations, dosages for adenovirus), 8,404,658 (formulations, dosages for AAV), and 5,846,946 (formulations, dosages for DNA plasmids), and publications related to clinical trials involving lentivirus, AAV, and adenovirus. For example, for AAV, the route of administration, formulation, and dosage can be as described in U.S. Patent No. 8,454,972 and clinical trials involving AAV. For adenovirus, the route of administration, formulation, and dosage can be as described in U.S. Patent No. 8,404,658 and clinical trials involving adenovirus.

[0286] For plasmid delivery, the route of administration, formulation, and dosage may be as described in U.S. Patent No. 5,846,946 and in clinical trials involving the plasmid. In certain embodiments, dosages may be estimated based on an average 70 kg individual (e.g., an adult male) and may be adjusted for patients, subjects, or mammals of different weights and species. The frequency of administration is within the skill of a medical or veterinary practitioner (e.g., physician, veterinarian), depending on routine factors including the patient's or subject's age, sex, overall health, other conditions, and the specific pathology or symptom being addressed. Viral vectors may be injected or otherwise delivered to the tissue or cells of interest.

[0287] With regard to in vivo delivery, AAV is advantageous over other viral vectors for several reasons, including its low toxicity (which may be due to a purification method that does not require ultracentrifugation of cellular particles that can activate an immune response) and its low potential for insertional mutagenesis because it does not integrate into the host genome.

[0288] The vectors and viral particles described herein can be delivered into host cells in vitro, in vivo, and / or ex vivo. Delivery can be achieved by any suitable method, including, but not limited to, physical, chemical, and biological methods. Physical delivery methods use physical forces to counteract the membrane barrier of a cell to facilitate intracellular delivery of the vector. Suitable physical methods include, but are not limited to, needles (e.g., injections), ballistic polynucleotide transfer (e.g., particle bombardment, microprojectile gene transfer, and gene guns), electroporation, sonoporation, photoporation, magnetofection, hydroporation, and mechanical massage. Chemical methods use chemicals to induce changes in cell membrane permeability or other properties that facilitate vector entry into the cell. For example, environmental pH can be changed, which can cause a change in cell membrane permeability. Biological methods rely on and utilize the biological processes or properties of the host cell that facilitate the transport of the vector into the cell (with or without a carrier). For example, the vector and / or its carrier may stimulate endocytosis or a similar process in the cell to facilitate uptake of the vector into the cell.

[0289] Delivery of engineered AAV capsid system components (e.g., engineered AAV capsids and / or polynucleotides encoding capsid proteins) to cells via particles. As used herein, the term "particle" refers to any particle of suitable size for delivery of the engineered AAV capsid system components described herein. Suitable sizes include macro-, micro-, and nano-sized particles. In certain embodiments, any of the engineered AAV capsid system components (e.g., polypeptides, polynucleotides, vectors, and combinations thereof described herein) can be bound, linked, integrated with, or otherwise associated with one or more particles or components thereof described herein. The particles described herein can then be administered to cells or organisms by an appropriate route and / or technique. In certain embodiments, particle delivery is selected for and may be advantageous for delivery of polynucleotide or vector components. It will be understood that in embodiments, particle delivery may also be advantageous for other engineered AAV capsid system molecules and formulations described elsewhere herein.

[0290] engineered viral particles containing engineered viral (e.g., AAV) capsids Also described herein are engineered viral particles (also referred to herein and elsewhere herein as "engineered viral particles") that can contain engineered viral capsids (e.g., AAV capsids, referred to as "engineered AAV capsids") as described in more detail elsewhere herein. It will be understood that an engineered AAV particle can be an adenovirus-based particle, a helper adenovirus-based particle, an AAV-based particle, or a hybrid adenovirus-based particle that contains at least one engineered AAV capsid protein as described above. An engineered AAV capsid is one that contains one or more engineered AAV capsid proteins as described elsewhere herein. In certain embodiments, an engineered AAV particle can contain between 1 and 60 engineered AAV capsid proteins as described herein. In some embodiments, an engineered AAV particle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, an engineered AAV particle may contain 0 to 59 wild-type AAV capsid proteins. In certain embodiments, an engineered AAV particle can contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type AAV capsid proteins. Thus, an engineered AAV particle can include one or more n-mer motifs as described above.

[0291] Engineered AAV particles can contain one or more cargo polynucleotides. Cargo polynucleotides are described in more detail elsewhere herein. Methods for producing engineered AAV particles from viral and non-viral vectors are described elsewhere herein. Formulations containing engineered viral particles are described elsewhere herein.

[0292] Cargo Polynucleotide Cargo is also described elsewhere herein. In some embodiments, cargo is a cargo polynucleotide that can be packaged into an engineered viral particle and then delivered to a cell. In some embodiments, delivery is muscle-specific. An engineered viral (e.g., AAV) capsid polynucleotide, other viral (e.g., AAV) polynucleotide, and / or vector polynucleotide can contain one or more cargo polynucleotides. In some embodiments, one or more cargo polynucleotides can be operably linked to an engineered viral (e.g., AAV) capsid polynucleotide and can be part of the engineered viral (e.g., AAV) genome of a viral (e.g., AAV) system of the present invention. The cargo polynucleotide can be packaged into an engineered viral (e.g., AAV) particle, which can be delivered to a cell, for example. In some embodiments, the cargo polynucleotide can be capable of modifying a polynucleotide (e.g., a gene or transcript) in a cell to which it is delivered. As used herein, "gene" can refer to a genetic unit that occupies a specific location on a chromosome and corresponds to a sequence of DNA that contains genetic instructions for a characteristic or trait in an organism. The term gene may refer to translated and / or untranslated regions of the genome. A "gene" may refer to a specific sequence of DNA that is translated into a polypeptide or transcribed into an RNA transcript, which may be a catalytic RNA molecule, including, but not limited to, tRNA, siRNA, piRNA, miRNA, long non-coding RNA, and shRNA. Modifications of polynucleotides, genes, transcripts, etc., include all genetic engineering techniques, including, but not limited to, gene editing and conventional recombinant genetic engineering techniques (e.g., total or partial gene insertion, deletion, and mutagenesis (e.g., insertion and deletion mutagenesis) techniques).

[0293] In some embodiments, the cargo molecule is a vaccine or a polynucleotide capable of encoding the same. In some embodiments, the vaccine can stimulate an immune response against cancer. In some embodiments, the vaccine can stimulate an immune response against colon cancer or pancreatic cancer. In some embodiments, the vaccine can create an environment that is hostile to hCG-producing cells, such as hCG-producing cancer cells.

[0294] Genetically modified cargo polynucleotides In some embodiments, the cargo molecule, whether delivered alone or as part of a system, can act to modify the genome, epigenome, and / or transcriptome of the cell to which it is delivered, regardless of whether it is delivered with other components of the system. Such systems include, but are not limited to, CRISPR-Cas systems. Other genetic engineering systems, such as TALENs, zinc finger nucleases, Cre-Lox, morpholinos, etc., are other non-limiting examples of genetic engineering systems in which one or more components can be delivered by the engineered viral (e.g., AAV) particles described herein.

[0295] In some embodiments, the cargo molecule is a gene editing system or a component thereof. In some embodiments, the cargo molecule is a CRISPR-Cas system molecule or a component thereof. In some embodiments, the cargo molecule is a polynucleotide encoding one or more components of a genetic modification system (such as a CRISPR-Cas system). In some embodiments, the cargo molecule is a gRNA.

[0296] In certain embodiments, the cargo molecule, when delivered alone or as part of a system, whether delivered with or without other components of the system, can be a polynucleotide or polypeptide that can act to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered so as to treat or prevent a muscular or skeletal disorder, a neurological disease or disorder, and / or a viral (such as a single-stranded RNA virus) disease, disorder, or symptom thereof. In certain embodiments, the cargo molecule, whether delivered with or without other components of the system, can act to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered so as to treat or prevent a premature aging disease (e.g., progeria laminopathies), glycogen storage disease, an immune disorder (such as an autoimmune disease), cancer, Duchenne muscular dystrophy (DMD), six limb-girdle muscular dystrophy disease (LGMD), Charcot-Marie-Tooth disease (CMT), MPS IIIA, Pompe disease, or other CNS-related diseases, such as Huntington's disease and other repeat expansion diseases.

[0297] In certain embodiments, the cargo molecule, whether delivered with other components of the system or not, acts to modify the genome, epigenome, and / or transcriptome of the cell to which it is delivered, such that it can modify the GAA gene, such as any of those described in U.S. Patent Application Publication No. 20190284555, the contents of which are incorporated by reference as if set forth herein in their entirety and which may be adapted for use with the present invention.

[0298] In some embodiments, the cargo molecule comprises an oligonucleotide linked to an MHCK7, CK8, or other muscle-specific promoter.

[0299] In some embodiments, the cargo molecule is a micro-dystrophin oligonucleotide containing only a selected region of the dystrophin gene optimized for protein functionality. In some embodiments, the selected region includes spectrin-like repeats 1, 2, 3, and 24. See, e.g., Harper SQ, Hauser MA, DelloRusso C, et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med. 2002;8(3):253-261. In some embodiments, the micro-dystrophin oligonucleotide is delivered by a rAAV agent known as AAVrh74.MHCK7 micro-dystrophin gene or SRP-9001, which is undergoing clinical trials NCT03375164 and NCT03769116. This micro-dystrophin gene construct contains NT-H1-R1-R2-R3-H2-R24-H4-CR-CT. In one embodiment, the microdystrophin gene comprises ABD-H1-R1-R2-R3-H2-R24-H4-CR-CT. In one embodiment, the microdystrophin gene comprises H, which represents the hinge region. England SB,et al.Nature.1990;343(6254):180-182;Wells DJ,et al.Hum Mol Genet.1995;4(8):1245-1250,Salva MZ,et al.Mol Ther.2007;15(2):320-329;Mendell JR,et al.Neurosci Lett.2012;527(2):90-99;Rodino-Klapac LR,et al.Hum Mol Genet.2013;22(24):4929-4937;Velazquez VM,et al.Mol Ther Methods Clin Dev.2017;4:159-168;Harper SQ,et al.Nat Med.2002;8(3):253-261;Nelson DM,et al.Hum Mol Genet.2018;27(12):2090-2100.In some embodiments, the selected region comprises at least spectrin-like repeats 2 and 3. In some embodiments, the microdystrophin gene comprises an nNOS domain. In some embodiments, the nNOS domain is composed of spectrin-like repeats 16 and / or 17. In some embodiments, the microdystrophin gene comprises spectrin-like repeats 16 and 17. In some embodiments, the nNOS domain is composed of spectrin-like repeats R1, R16, R17, R23, and R24. In some embodiments, the microdystrophin gene is linked to a muscle-specific promoter. In some embodiments, the microdystrophin oligonucleotide is linked to MHCK7, CK8, SNP18, SP0033, SP0051, SP0173, tmCK, or another muscle-specific promoter.

[0300] In one embodiment, the cargo microdystrophin comprises an ABD (actin-binding domain), one or more hinge regions (e.g., H1, H2, H3, H4), and one or more spectrin-like repeats (e.g., R1, R1' R2, R3, R16, R17, R20, R21, R22, R23, R24, R24', and optionally a dystroglycan binding domain (DBD). In some embodiments, micro-dystrophin is composed of ABD-H1-R1-R16-R17-R23-R24-H4-DBD. In some embodiments, micro-dystrophin is composed of ABD-H1-R1-R2-R3-H2-R24-H4-CR. In some embodiments, the micro-dystrophin gene comprises ABD-H1-R1-R2-R3-H2-R24-H4-CR-CT. In some embodiments, the micro-dystrophin gene comprises ABD-H1-R1'-R24'-H4-CR-CT.

[0301] In some embodiments, the cargo molecule is a polynucleotide capable of encoding a microdystrophin gene, wherein the microdystrophin gene contains spectrin-like repeats R1, R16, R17, R23, and R24. In some embodiments, the microdystrophin gene contains hinge region (H)4 and / or H1. In some embodiments, the microdystrophin gene contains an N-terminal actin-binding domain. In some embodiments, the microdystrophin gene contains the C-terminal dystroglycan-binding domain of human full-length dystrophin protein. The microdystrophin gene may contain an nNOS domain. In some embodiments, the nNOS domain is composed of spectrin-like repeats 16 and / or 17. In some embodiments, the microdystrophin gene comprises spectrin-like repeats 16 and 17. The micro-dystrophin gene may be as described in WO2019118806A1 and WO2016 / 115543, which are incorporated by reference as if set forth herein in their entirety and may be adapted for use with the present invention. In one embodiment, the cargo polynucleotide may encode a 5-repeat micro-dystrophin protein containing, from N- to C-terminus, the N-terminal actin-binding domain, hinge region 1 (H1), spectrin-like repeats R1, R16, R17, R23, and R24, hinge region 4 (H4), and a C-terminal dystroglycan-binding domain of the human full-length dystrophin protein. The protein sequences of this 5-repeat micro-dystrophin and related dystrophin minigenes are described in WO2016 / 115543. In one embodiment, the cargo polynucleotide may correspond to the microdystrophin gene, which is part of a drug known as SGT001, currently in clinical trials with the identification number NCT03368742.

[0302] In some embodiments, the cargo molecule is a minidis gene or vector, which may be composed of ABD-H1-R1-R2-R3-R16-R17-H3-R20-R21; ABD-H1-R1-R2-R3-R16-R17-H3-R20-R21-R22-R23-R24-H4-CR; or H3-R20-R21-R22-R23-R24-H4-CR-CT.

[0303] In some embodiments, the cargo molecule is SCGB cDNA. In some embodiments, the SGCB cDNA is linked to an MHCK7, CK8 promoter, SNP18 promoter, SP0033 promoter, SP0051, SP0173 promoter, tmCK promoter, or another muscle-specific promoter. In some embodiments, the cargo molecule is β-sarcoglycan cDNA, α-sarcoglycan cDNA, dysferlin cDNA, γ-sarcoglycan cDNA, calpin-3 cDNA, SGSH cDNA (e.g., LYS-SAF302), neurotropin 3 cDNA, anoctamin-5 cDNA, or any combination thereof.

[0304] In certain embodiments, the cargo molecule, whether delivered along with other components of the system or not, acts to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered so as to treat, prevent, and / or modify a gene or gene product associated with a repeat expansion disease, such as Huntington's disease, for example, those described in U.S. Patent Application Publication No. 20190100755, U.S. Patent No. 10,066,228, the contents of which are incorporated by reference as if set forth herein in their entirety and may be adapted for use with the present invention.

[0305] In certain embodiments, the cargo molecule is an antisense oligomer or RNA molecule, such as those disclosed in U.S. Patent Application Publication Nos. 20160251398, 20150267202, 20190015440, 20140287983, 20180216111, and International Patent Application Publication No. WO 20160251398, the contents of which are incorporated by reference as if set forth herein in their entirety and may be adapted for use with the present invention. Those disclosed in U.S. Patent Application Publication No. 2017 / 062835, U.S. Patent Application Publication No. 20190177723, U.S. Patent Application Publication No. 20170051278, U.S. Patent Application Publication No. 20180271893, WO 2016 / 14965, U.S. Patent No. 10,076,536, WO 2018 / 00580, WO 2018 / 11866, and WO 2019 / 059973.

[0306] In certain embodiments, the cargo molecule, whether delivered with or without other components of the system, acts to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered, such that it treats or prevents single-stranded RNA viruses, e.g., influenza, West Nile virus, SARS, hepatitis C, dengue fever, Ebola, Marburg, and / or calicivirus. In certain embodiments, the cargo molecule can be an antisense antiviral compound, such as any of those described in U.S. Pat. No. 8,703,735 B2, the contents of which are incorporated by reference as if set forth herein in their entirety, and which may be adapted for use with the present invention.

[0307] Further exemplary genetic and gene-associated diseases and genes that can be modified by the cargo molecules described herein are listed elsewhere herein, see, for example, Tables 5-6.

[0308] In some embodiments, the cargo molecule may add or modify the GALGT2 gene. Instead of acting to replace missing dystrophin, GALGT2 gene therapy enhances the structural integrity of muscle in a way that compensates for the absence of dystrophin by increasing the expression of a protein that is neither mutated nor missing in the disease. GALGT2 offers the potential to treat DMD regardless of the specific dystrophin mutation, and also has utility in other muscular dystrophies.

[0309] In some embodiments, the cargo molecule is a morpholino, such as those described in U.S. Patent Application Publication Nos. 2018 / 0161359 and 2019 / 0054113, the contents of which are incorporated by reference as if set forth herein in their entirety and may be adapted for use with the present invention. In some embodiments, the morpholino is a morpholino oligomer (PMO) or a peptide-linked morpholino (PPMO). PMO-based platforms can be used to treat genetic diseases by altering mRNA transcription. PMOs are synthetic chemical structures modeled after the natural framework of RNA. While PMOs have the same nucleobases found in RNA, they are linked to a hexagonal morpholine ring instead of a pentagonal ribose ring. Furthermore, the morpholine rings are connected to each other by phosphorodiamidate bonds instead of the phosphodiester bonds found in RNA. PMOs and PPMOs can be used for exon skipping and translational repression.

[0310] In certain embodiments, the cargo molecule may be a peptide-oligomer conjugate, for example, as described in International Patent Application Publication No. WO 2017106304 A1, the contents of which are incorporated by reference as if set forth herein in their entirety, and which may be adapted for use with the present invention.

[0311] In some embodiments, the morpholino is the morpholino found in eteplirsen, which may be effective for targeting exon 51 of the dystrophin mRNA. In some embodiments, the cargo molecule may produce exon skipping in the context of DMD, such as those described in U.S. Patent Application Publication Nos. 2014 / 0315977A1 ​​and 2018 / 010581, the contents of which are incorporated by reference as if set forth herein in their entirety and may be adapted for use with the present invention.

[0312] Exon skipping In some embodiments, the nucleotide sequence may encode a nucleic acid capable of inducing exon skipping. Such an encoded nucleic acid may be an antisense oligonucleotide or an antisense nucleotide system. As used herein, the term "exon skipping" refers to the modification of pre-mRNA splicing by targeting splice donor and / or acceptor sites within the pre-mRNA with one or more complementary antisense oligonucleotides (AONs). By blocking spliceosome access to one or more splice donor or acceptor sites, AONs can prevent the splicing reaction, thereby causing the deletion of one or more exons from the fully processed mRNA. Exon skipping can be achieved in the nucleus during the maturation process of pre-mRNA. In some instances, exon skipping can involve masking key sequences involved in the splicing of targeted exons by using antisense oligonucleotides (AONs) complementary to splice donor sequences within the pre-mRNA.

[0313] In one embodiment, the nucleotide sequence encodes an antisense oligonucleotide or antisense nucleotide system capable of inducing exon skipping in dystrophin mRNA. For example, a nonsense or frameshift mutation within exon x of the dystrophin gene results in a carboxy-terminally truncated, non-functional dystrophin protein. Expression of the mature mRNA transcript can result in a functional dystrophin protein that lacks the amino acids encoded by exon x but contains dystrophin amino acids both N- and C-terminal to the deleted amino acids.

[0314] The nucleotide sequence is for exons 1, 2, 3, 4, 5, 6, 7, 8, 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, 45, 36, 37, 38, 39, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 The nucleotide sequence may encode an antisense oligonucleotide or antisense nucleotide system capable of inducing exon skipping at exons 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or any combination thereof. The nucleotide sequence may encode an antisense oligonucleotide or antisense nucleotide system capable of inducing exon skipping at exons 43, 44, 50, 51, 52, 55, or any combination thereof.

[0315] CRISPR-Cas system cargo molecules In some embodiments, an engineered virus (e.g., AAV) or other particle described herein can include one or more CRISPR-Cas system molecules, which can be polynucleotides or polypeptides. In some embodiments, a polynucleotide can encode one or more CRISPR-Cas system molecules. In some embodiments, a polynucleotide can encode a Cas protein, a CRISPR cascade protein, a gRNA, or a combination thereof. Other CRISPR-Cas system molecules are described elsewhere herein and can be delivered as either polypeptides or polynucleotides.

[0316] Generally, CRISPR-Cas or CRISPR system, as used herein and in the literature, such as International Patent Application Publication No. WO 2014 / 093622 (PCT / US2013 / 074667), refers collectively to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" with respect to endogenous CRISPR systems), or "RNAs" as that term is used herein (e.g., RNAs to guide Cas, such as Cas9, e.g., CRISPR CRISPR systems include RNA and transactivating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from the CRISPR locus. Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence (also called a protospacer in the context of endogenous CRISPR systems). See, e.g., Shmakov et al. (2015) "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems," Molecular Cell, DOI: dx.doi.org / 10.1016 / j.molcel.2015.10.008.

[0317] In certain embodiments, a protospacer adjacent motif (PAM) or PAM-like motif directs binding of the effector protein complex disclosed herein to a target locus of interest. In certain embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer). The term "PAM" can be used interchangeably with the terms "PFS" or "protospacer adjacent site" or "protospacer adjacent sequence."

[0318] In a preferred embodiment, the CRISPR effector protein can recognize a 3' PAM. In a specific embodiment, the CRISPR effector protein can recognize a 3' PAM that is a 5' H (where H is A, C, or U).

[0319] With respect to the formation of a CRISPR complex, the term "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence may comprise an RNA polynucleotide. The term "target RNA" refers to an RNA polynucleotide that is or contains a target sequence. In other words, the target RNA may be a portion of a gRNA, i.e., an RNA polynucleotide or a portion of an RNA polynucleotide to which a guide sequence is designed to have complementarity and to which an effector function mediated by a complex comprising a CRISPR effector protein and a gRNA is directed. In certain embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0320] In some exemplary embodiments, the CRISPR effector protein can be delivered using a nucleic acid molecule encoding the CRISPR effector protein. The nucleic acid molecule encoding the CRISPR effector protein can advantageously be a codon-optimized CRISPR effector protein. An example of a codon-optimized sequence is a sequence optimized for expression in a eukaryote, such as a human (i.e., optimized for expression in a human), or a sequence optimized for another eukaryote, animal, or mammal as described herein; see, for example, the SaCas9 human codon-optimized sequence in International Patent Application Publication No. WO 2014 / 093622 (PCT / US2013 / 074667). While this is preferred, it is understood that other examples are possible, and codon optimization for host species other than humans, or for specific organs, is known. In some embodiments, the enzyme coding sequence encoding the CRISPR effector protein is codon-optimized for expression in a specific cell, such as a eukaryotic cell. Eukaryotic cells may be of or derived from specific organisms, such as plants or mammals, including, but not limited to, humans, or non-human eukaryotic organisms or animals or mammals described herein, such as mice, rats, rabbits, dogs, livestock, or human mammals or primates. In certain embodiments, processes for modifying the germline genetic identity of humans and / or processes for modifying the genetic identity of animals that may cause suffering to humans or animals without substantial medical benefit, as well as animals resulting from such processes, may also be excluded. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon (e.g., about or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon more frequently or most frequently used in the genes of that host cell, while maintaining the native amino acid sequence. Different species exhibit specific biases for particular codons of particular amino acids.Codon bias (differences in codon usage among organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The prevalence of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database," available at kazusa.orjp / codon / , and these tables can be adapted in several ways. See Nakamura, Y., et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a Cas-encoding sequence correspond to the most frequently used codon for a particular amino acid.

[0321] In certain embodiments, the methods described herein may include providing a Cas transgenic cell in which one or more nucleic acids encoding one or more guide RNAs are provided or introduced into the cell in operably linked relationship to regulatory elements, including promoters of one or more genes of interest. As used herein, the term "Cas transgenic cell" refers to a cell, such as a eukaryotic cell, into which a Cas gene has been genomically integrated. The nature, type, or origin of the cell is not particularly limited according to the present invention. Furthermore, the method by which the Cas transgene is introduced into the cell may vary and may be any method known in the art. In certain embodiments, the Cas transgenic cell is obtained by introducing the Cas transgene into an isolated cell. In certain other embodiments, the Cas transgenic cell is obtained by isolating cells from a Cas transgenic organism. By way of example, and not limitation, the Cas transgenic cell referred to herein may be obtained from a Cas transgenic eukaryotic organism, such as a Cas knock-in eukaryotic organism. See International Patent Application Publication No. WO 2014 / 093622 (PCT / US13 / 74667), which is incorporated herein by reference. U.S. Patent Application Publication Nos. 20120017290 and 20110265198, assigned to Sangamo BioSciences, Inc., for methods targeting the Rosa locus, can be modified to use the CRISPR Cas system of the present invention. U.S. Patent Application Publication No. 20130236946, assigned to Cellectis, for methods targeting the Rosa locus, can also be modified to use the CRISPR Cas system of the present invention. As a further example, see Platt et al. (Cell; 159(2):440-455 (2014)), which describes a Cas9 knock-in mouse, which is incorporated herein by reference. The Cas transgene further contains a Lox-Stop-PolyA-Lox (LSL) cassette, which allows Cas expression to be inducible by Cre recombinase.Alternatively, Cas transgenic cells may be obtained by introducing a Cas transgene into isolated cells. Delivery systems for transgenes are well known in the art. For example, Cas transgenes can be delivered to eukaryotic cells, for example, by vectors (e.g., AAV, adenovirus, lentivirus) and / or particle and / or nanoparticle delivery, as similarly described elsewhere herein. Lentiviral and retroviral systems, as well as non-viral systems, for delivering CRISPR-Cas system components are generally known in the art. AAV- and adenovirus-based systems for CRISPR-Cas system components are generally known in the art and are also described herein (e.g., the engineered AAV of the present invention).

[0322] It will be understood by those skilled in the art that cells such as the Cas transgenic cells referred to herein may contain additional genomic alterations in addition to having mutations that result from the sequence-specific action of Cas when complexed with an integrated Cas gene or an RNA capable of guiding Cas to a target locus.

[0323] In certain embodiments, the present invention includes vectors for, for example, delivering or introducing Cas and / or RNA capable of guiding Cas to a target locus (i.e., guide RNA) into a cell, but also for propagating these components (e.g., in a prokaryotic cell). This may be in addition to delivery of one or more CRISPR-Cas components or other genetic recombination system components not already delivered by the engineered AAV particles described herein. As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to result in replication of the inserted segment. Generally, vectors are capable of replication when associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends or no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome.Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0324] A recombinant expression vector can contain the nucleic acid of the present invention in a form suitable for expression in a host cell, which means that the recombinant expression vector contains one or more regulatory elements that can be selected based on the host cell used for expression, operably linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). Regarding recombination and cloning methods, reference is made to U.S. Patent Application Publication No. 2004 / 0171156, the entire contents of which are incorporated herein by reference. Thus, the embodiments disclosed herein can also include transgenic cells that contain a CRISPR effector system. In some exemplary embodiments, transgenic cells can function as individual, separate volumes. In other words, a sample containing the masking construct may be delivered to a cell, for example in a suitable delivery vesicle, and if the target is present in the delivery vesicle, the CRISPR effector is activated and a detectable signal is generated.

[0325] The vector may include regulatory elements, such as a promoter. The vector may include a Cas coding sequence and / or may also include a single, and optionally at least 3, 8, 16, 32, 48, or 50 guide RNA (e.g., sgRNA) coding sequence, e.g., 1-2, 1-3, 1-4, 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, or 3-50 RNAs (e.g., sgRNAs). Advantageously, if up to about 16 RNAs are present in a single vector, there may be a promoter per RNA (e.g., sgRNA); if a single vector provides more than 16 RNAs, one or more promoters may drive expression of two or more of the RNAs, e.g., if 32 RNAs are present, each promoter may drive expression of two RNAs, and if 48 RNAs are present, each promoter may drive expression of three RNAs. With simple calculations, well-established cloning protocols, and the teachings of this disclosure, one skilled in the art can easily implement the present invention for RNA for a suitable exemplary vector, such as AAV, and a suitable promoter, such as the U6 promoter. For example, the packaging limit of AAV is approximately 4.7 kb. The length of a single U6-gRNA (and restriction sites for cloning) is 361 bp. Therefore, one skilled in the art can easily place approximately 12 to 16, e.g., 13, U6-gRNA cassettes into a single vector, which can be assembled by any suitable means, such as the golden gate strategy used in TALE assembly (genome-engineering.org / taleffectors / ). One skilled in the art can also use the tandem guide method to increase the number of U6-gRNAs by approximately 1.5-fold, e.g., from 12 to 16, e.g., 13, to approximately 18 to 24, e.g., approximately 19, U6-gRNAs. Thus, one skilled in the art can easily arrive at about 18 to 24, e.g., about 19 promoter-RNAs, e.g., U6-gRNAs, in a single vector, e.g., an AAV vector.Another way to increase the number of promoters and RNAs in a vector is to use a single promoter (e.g., U6) to express an array of RNAs separated by cleavable sequences. Yet another way to increase the number of promoter-RNAs in a vector is to express promoter-RNAs separated by cleavable sequences in the coding sequence or intron of a gene; in this case, it is advantageous to use a polymerase II promoter, which can result in increased expression and enable tissue-specific transcription of long RNAs. (See, for example, nar.oxfordjournals.org / content / 34 / 7 / e53.short and nature.com / mt / journal / v16 / n9 / abs / mt2008144a.html). In an advantageous embodiment, an AAV can package U6 tandem gRNAs targeting up to about 50 genes. Thus, from knowledge in the art and from the teachings in this disclosure, one of skill in the art can readily make and use vectors, e.g., a single vector, that express multiple RNAs or guides under the control of, or operably or functionally linked to, one or more promoters without undue experimentation, particularly with respect to the number of RNAs or guides described herein.

[0326] The guide RNA coding sequence and / or the Cas coding sequence can be functionally or operably linked to a regulatory element, which drives expression. The promoter can be a constitutive promoter, a conditional promoter, an inducible promoter, and / or a tissue-specific promoter. The promoter can be selected from the group consisting of RNA polymerase, pol I, pol II, pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. A preferred promoter is U6.

[0327] Additional effectors for use in the present invention can be identified by their proximity to the Cas1 gene, for example, but not limited to, within the region 20 kb from the start of the Cas1 gene and 20 kb from the end of the Cas1 gene. In certain embodiments, the effector protein comprises at least one HEPN domain and at least 500 amino acids, and the C2c2 effector protein is naturally present in the prokaryotic genome within 20 kb upstream or downstream of the Cas gene or CRISPR array. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas12a, Cas13a, Cas13b, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csx1, Csx ... Examples of C2c2 effector proteins include sa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. In some exemplary embodiments, the C2c2 effector protein is naturally present within 20 kb upstream or downstream of the Cas1 gene in a prokaryotic genome. The terms "orthologue" (also referred to herein as "ortholog") and "homologue" (also referred to herein as "homolog") are well known in the art. By further guidance, a "homolog" of a protein, as used herein, is a protein of the same species that performs the same or similar function as the protein to which it is a homolog. Homologous proteins may, but are not necessarily, structurally related, or are only partially structurally related. An "ortholog" of a protein, as used herein, is a protein of a different species that performs the same or similar function as the protein to which it is an ortholog.Orthologous proteins may, but are not necessarily, structurally related, or are only partially structurally related.

[0328] In certain embodiments, one or more elements of the nucleic acid targeting system are derived from a specific organism that contains an endogenous CRISPR RNA targeting system. In certain embodiments, the CRISPR RNA targeting system is found in the genera Eubacterium and Ruminococcus. In certain embodiments, the effector protein contains targeted collateral ssRNA cleavage activity. In certain embodiments, the effector protein contains a dual HEPN domain. In certain embodiments, the effector protein lacks the corresponding Helical-1 domain of Cas13a. In certain embodiments, the effector protein is smaller than previously characterized Class 2 CRISPR effectors, with a median size of 928 aa. This median size is 190 aa (17%) smaller than that of Cas13c, over 200 aa (18%) smaller than that of Cas13b, and over 300 aa (26%) smaller than that of Cas13a. In certain embodiments, the effector protein does not require flanking sequences (eg, PFS, PAM).

[0329] In certain embodiments, the effector protein locus structure comprises a WYL domain-containing accessory protein (designated after the three amino acids conserved in the first identified group of these domains; see, e.g., WYL domain IPR026881). In certain embodiments, the WYL domain accessory protein comprises at least one helix-turn-helix (HTH) or ribbon-helix-helix (RHH) DNA-binding domain. In certain embodiments, the WYL domain-containing accessory protein increases both the targeted and collateral ssRNA cleavage activity of the RNA-targeting effector protein. In certain embodiments, the WYL domain-containing accessory protein comprises an N-terminal RHH domain and a pattern of primarily hydrophobic conserved residues, including an invariant tyrosine-leucine doublet corresponding to the original WYL motif. In certain embodiments, the WYL domain-containing accessory protein is WYL1. WYL1 is a single WYL domain protein primarily associated with the genus Ruminococcus.

[0330] In another exemplary embodiment, the Type VI RNA-targeting Cas enzyme is Cas13d. In a specific embodiment, Cas13d is Eubacterium siraeum DSM 15702 (EsCas13d) or Ruminococcus sp. N15.MGS-57 (RspCas13d) (see, e.g., Yan et al., "Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein," Molecular Cell (2018), doi.org / 10.1016 / j.molcel.2018.02.028). RspCas13d and EsCas13d do not require flanking sequences (e.g., PFS, PAM).

[0331] The methods, systems, and tools provided herein can be designed for use with Class 1 CRISPR proteins, which can be Type I, Type III, or Type IV Cas proteins, as described in Makarova et al., The CRISPR Journal, v. 1, n., 5 (2018); DOI: 10.1089 / crispr.2018.0033, which is incorporated herein by reference in its entirety, and in particular as described in Figure 1, p. 326. Class 1 systems typically use multiprotein effector complexes, which in some embodiments can include auxiliary proteins, e.g., one or more proteins in a complex referred to as a CRISPR-associated complex for antiviral defense (cascade), one or more adaptation proteins (e.g., Cas1, Cas2, RNA nuclease), and / or one or more accessory proteins (e.g., Cas4, DNA nuclease), a CRISPR-associated Rossmann fold (CARF) domain-containing protein, and / or an RNA transcriptase. Although class 1 systems share limited sequence similarity, class 1 system proteins can be identified by their similar structure, including one or more subunits of the repeat-associated mysterious protein (RAMP) family, such as Cas5, Cas6, and Cas7. RAMP proteins are characterized by having one or more RNA recognition motif domains. The large subunit (e.g., Cas8 or Cas10) and small subunit (e.g., Cas11) are also unique to class 1 systems. See, e.g., Figures 1 and 2. Koonin EV, Makarova KS. 2019 Origins and evolution of CRISPR-Cas systems. Phil. Trans. R. Soc. B 374:20180087, DOI: 10.1098 / rstb.2018.0087. In one embodiment, class 1 systems are characterized by the signature protein Cas3. A cascade in a particular Class 1 protein may comprise a dedicated complex of multiple Cas proteins that bind to the pre-crRNA and recruit additional Cas proteins, e.g., Cas6 or Cas5, which are nucleases directly involved in processing the pre-crRNA.In one embodiment, type I CRISPR proteins comprise an effector complex containing one or more Cas5 subunits and two or more Cas7 subunits. Class 1 subtypes include types IA, IB, IC, IU, ID, IE, and IF, types IV-A and IV-B, and types III-A, III-D, III-C, and III-B. Class 1 systems also include CRISPR-Cas variants, including type IA, IB, IE, IF, and IU variants, which may include transposon- and plasmid-borne variants, including versions of subtype IF encoded by the large family of Tn7-like transposons and a smaller group of Tn7-like transposons encoding similarly resolved subtype IB systems. See also Peters et al., PNAS 114(35)(2017); DOI:10.1073 / pnas.1709035114; Makarova et al., The CRISPR Journal, v.1, n5, Figure 5.

[0332] Cas molecule In some embodiments, the cargo molecule may be or comprise a Cas polypeptide and / or polynucleotide capable of encoding a Cas polypeptide or a fragment thereof. Any Cas molecule may be a cargo molecule. In some embodiments, the cargo molecule is a Class I CRISPR-Cas system Cas polypeptide. In some embodiments, the cargo molecule is a Class II CRISPR-Cas system Cas polypeptide. In some embodiments, the Cas polypeptide is a Type I Cas polypeptide. In some embodiments, the Cas polypeptide is a Type II Cas polypeptide. In some embodiments, the Cas polypeptide is a Type III Cas polypeptide. In some embodiments, the Cas polypeptide is a Type IV Cas polypeptide. In some embodiments, the Cas polypeptide is a Type V Cas polypeptide. In some embodiments, the Cas polypeptide is a Type VI Cas polypeptide. In some embodiments, the Cas polypeptide is a Type VII Cas polypeptide. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas12a, Cas13a, Cas13b, Cas13c, Cas13d, Csy1, Csy2, Csy3, Cse1, Cse2, and Csc1. , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof.

[0333] Guide Sequence As used herein, the terms "guide sequence" and "guide molecule" in the context of the CRISPR-Cas system include any polynucleotide sequence that has sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid targeting complex to the target nucleic acid sequence. Guide sequences generated using the methods disclosed herein can be full-length guide sequences, truncated guide sequences, full-length sgRNA sequences, truncated sgRNA sequences, or E+F sgRNA sequences. Each gRNA can be designed to contain multiple binding recognition sites (e.g., aptamers) specific for the same or different adapter proteins. Each gRNA can be designed to bind to the promoter region -1000 to +1 nucleic acid, preferably -200 nucleic acid, upstream of the transcription start site (i.e., TSS). This positioning improves functional domains that affect gene activation (e.g., transcriptional activators) or gene inhibition (e.g., transcriptional repressors). The modified gRNA can be one or more modified gRNAs (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs) that target one or more target loci included in the composition. The multiple gRNA sequences can be arranged in tandem, preferably separated by direct repeats.

[0334] In certain embodiments, the degree of complementarity of a guide sequence to a given target sequence, when optimally aligned using a suitable alignment algorithm, can be about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some exemplary embodiments, a guide molecule includes a guide sequence that can be designed to have at least one mismatch with the target sequence so that an RNA duplex is formed between the guide sequence and the target sequence. Thus, the degree of complementarity is preferably less than 99%. For example, if the guide sequence consists of 24 nucleotides, the degree of complementarity is more specifically about 96% or less. In certain embodiments, the guide sequence is designed to have a stretch of two or more adjacent mismatched nucleotides, so that the degree of complementarity across the entire guide sequence is further reduced. For example, if the guide sequence consists of 24 nucleotides, the degree of complementarity is more particularly about 96% or less, more particularly about 92% or less, more particularly about 88% or less, more particularly about 84% or less, more particularly about 80% or less, more particularly about 76% or less, and more particularly about 72% or less, depending on whether the stretch of two or more mismatched nucleotides includes 2, 3, 4, 5, 6, or 7 nucleotides, etc. In certain embodiments, besides the stretch of one or more mismatched nucleotides, the degree of complementarity is about or about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, greater than 99%, or more when optimally aligned using a suitable alignment algorithm.Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence can be assessed by any suitable assay. For example, sufficient components of a nucleic acid-targeting CRISPR system to form a nucleic acid-targeting complex containing the guide sequence to be tested can be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with a vector encoding the components of the nucleic acid-targeting complex, followed by evaluation of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by a Surveyor assay described herein. Similarly, cleavage of a target nucleic acid sequence (or a sequence nearby) can be evaluated in a test tube by providing components of a nucleic acid-targeting complex containing the target nucleic acid sequence, the guide sequence to be tested, and a control guide sequence that differs from the test guide sequence, and comparing the rate of binding or cleavage at or near the target sequence between the test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art. Guide sequences, and thus nucleic acid-targeting guide RNAs, can be selected to target any target nucleic acid sequence.

[0335] As used herein, the terms "crRNA" or "guide RNA" or "single guide RNA" or "sgRNA" or "one or more nucleic acid components" of a Type V or Type VI CRISPR-Cas locus effector protein include any polynucleotide sequence that has sufficient complementarity with a target nucleic acid sequence to hybridize with and direct sequence-specific binding of a nucleic acid targeting complex to the target nucleic acid sequence. In certain embodiments, the degree of complementarity is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence can be assessed by any suitable assay. For example, components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex containing the guide sequence to be tested can be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with a vector encoding the components of the nucleic acid-targeting complex, followed by assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by a Surveyor assay described herein.Similarly, cleavage of a target nucleic acid sequence can be assessed in a test tube by providing components of a nucleic acid targeting complex that include the target nucleic acid sequence, a guide sequence to be tested, and a control guide sequence that differs from the test guide sequence, and comparing the rate of binding or cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art. The guide sequence, and thus the nucleic acid targeting guide, can be selected to target any target nucleic acid sequence. The target sequence can be DNA. The target sequence can be any RNA sequence. In certain embodiments, the target sequence can be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double-stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmic RNA (scRNA). In a preferred embodiment, the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In a preferred embodiment, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA and lncRNA. In a more preferred embodiment, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.

[0336] In some embodiments, the nucleic acid targeting guide is selected to reduce the degree of secondary structure within the nucleic acid targeting guide. In some embodiments, less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less of the nucleotides in the nucleic acid targeting guide participate in self-complementary base pairing when optimally folded. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online web server RNAfold developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, e.g., A.R. Gruber et al., 2008, Cell 106(1):23-24; and P.A. Carr and G.M. Church, 2009, Nature Biotechnology 27(12):1151-62).

[0337] In certain embodiments, a guide RNA or crRNA can comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide or spacer sequence. In certain embodiments, a guide RNA or crRNA can comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide or spacer sequence. In certain embodiments, the direct repeat sequence can be located upstream (i.e., 5') of the guide or spacer sequence. In other embodiments, the direct repeat sequence can be located downstream (i.e., 3') of the guide or spacer sequence.

[0338] In certain embodiments, the crRNA comprises a stem-loop, preferably a single stem-loop. In certain embodiments, the direct repeat sequence forms a stem-loop, preferably a single stem-loop.

[0339] In certain embodiments, the spacer length of the guide RNA is 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is 15 to 17 nt, for example, 15, 16, or 17 nt, 17 to 20 nt, for example, 17, 18, 19, or 20 nt, 20 to 24 nt, for example, 20, 21, 22, 23, or 24 nt, 23 to 25 nt, for example, 23, 24, or 25 nt, 24 to 27 nt, for example, 24, 25, 26, or 27 nt, 27 to 30 nt, for example, 27, 28, 29, or 30 nt, 30 to 35 nt, for example, 30, 31, 32, 33, 34, or 35 nt, or 35 nt or more.

[0340] The "tracrRNA" sequence or similar term includes any polynucleotide sequence that has sufficient complementarity with the crRNA sequence to hybridize. In certain embodiments, the degree of complementarity between the tracrRNA sequence and the crRNA sequence along the length of the shorter of the two when optimally aligned is about or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more. In certain embodiments, the tracr sequence is about or greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In certain embodiments, the tracr sequence and the crRNA sequence are contained within a single transcript such that hybridization between the two produces a transcript with secondary structure, such as a hairpin. In one embodiment of the present invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In a preferred embodiment, the transcript has two, three, four, or five hairpins. In a further embodiment of the present invention, the transcript has up to five hairpins. In the hairpin structure, the portion of the sequence 5' of the final "N" and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3' of the loop corresponds to the tracr sequence.

[0341] Generally, the degree of complementarity is along the shorter length of the two sequences with respect to optimal alignment of the sca and tracr sequences. Optimal alignment may be determined by any suitable alignment algorithm and may further account for secondary structure, such as self-complementarity, in either the sca or tracr sequences. In certain embodiments, the degree of complementarity between the tracr and sca sequences along the shorter length of the two sequences when optimally aligned is about or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, greater than 99%, or greater.

[0342] Generally, CRISPR-Cas, CRISPR-Cas9, or CRISPR system, as used in the above-mentioned documents, such as International Patent Application Publication No. WO 2014 / 093622 (PCT / US2013 / 074667), collectively refers to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, particularly, in the case of CRISPR-Cas9, sequences encoding the Cas9 gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" with respect to endogenous CRISPR systems), or "RNAs" as that term is used herein (e.g., RNAs that guide Cas9, e.g., CRISPR These include RNA and transactivating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA), or other sequences and transcripts from a CRISPR locus. Generally, CRISPR systems feature elements that promote the formation of a CRISPR complex at the site of a target sequence (also called a protospacer in the context of endogenous CRISPR systems). With respect to the formation of a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to be complementary, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The section of a guide sequence whose complementarity to the target sequence is important for cleavage activity is referred to herein as a seed sequence. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell, and can include nucleic acids within or derived from mitochondria, organelles, vesicles, liposomes, or particles present within the cell. In some embodiments, particularly for non-nuclear use, NLSs are not preferred. In some embodiments, a CRISPR system includes one or more nuclear export signals (NESs).In some embodiments, the CRISPR system comprises one or more NLSs and one or more NESs. In some embodiments, direct repeats can be identified in silico by searching for repetitive motifs that meet any or all of the following criteria: 1. found in a 2 Kb window of genomic sequence adjacent to the type II CRISPR locus; 2. spanning 20-50 bp; and 3. spaced 20-50 bp apart. In some embodiments, two of these criteria can be used, for example, 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used.

[0343] In embodiments of the present invention, the terms guide sequence and guide RNA, i.e., RNA capable of guiding Cas to a target genomic locus, are used interchangeably as in the above-cited references, such as International Patent Application Publication No. WO 2014 / 093622 (PCT / US2013 / 074667). Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In certain embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably, the guide sequence is 10-30 nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay.For example, components of a CRISPR system sufficient to form a CRISPR complex containing a guide sequence to be tested can be provided to a host cell containing the corresponding target sequence, such as by transfection with a vector encoding the CRISPR sequence components, followed by assessment of preferential cleavage within the target sequence, such as by a Surveyor assay described herein. Similarly, cleavage of a target polynucleotide sequence can be assessed in a test tube by providing components of a CRISPR complex containing the target sequence, the guide sequence to be tested, and a control guide sequence that differs from the test guide sequence, and comparing the rate of binding or cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art.

[0344] In certain embodiments of the CRISPR-Cas system, the degree of complementarity between a guide sequence and its corresponding target sequence can be about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; the guide or RNA or sgRNA can be about or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or the guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length; advantageously, the tracr RNA is 30 or 50 nucleotides in length. However, one embodiment of the present invention is directed to reducing off-target interactions, e.g., reducing guide interactions with target sequences with low complementarity. Indeed, examples show that the invention includes mutations that result in a CRISPR-Cas system that can distinguish between target sequences and off-target sequences with greater than 80% to about 95% complementarity, e.g., 83% to 84%, or 88 to 89%, or 94 to 95% complementarity (e.g., distinguishing between a target with 18 nucleotides and an 18 nucleotide off-target with one, two, or three mismatches). Thus, for the present invention, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100%.An off-target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, advantageously an off-target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide.

[0345] In particularly preferred embodiments of the present invention, the guide RNA (capable of guiding Cas to a target locus) can include (1) a guide sequence capable of hybridizing to a genomic target locus in a eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All of (1) through (3) can be present in a single RNA, i.e., sgRNA (arranged in a 5' to 3' orientation), or the tracr RNA can be a separate RNA from the RNA containing the guide and tracr sequences. The tracr hybridizes to the tracr mate sequence and directs the CRISPR / Cas complex to the target sequence. When the tracr RNA is present in a separate RNA from the RNA containing the guide and tracr sequences, the length of each RNA can be optimized to be shorter than their respective native lengths, and each can be independently chemically modified to protect against degradation by cellular RNases or otherwise increase stability.

[0346] The methods of the invention described herein include inducing one or more mutations in a eukaryotic cell (in vitro, i.e., in an isolated eukaryotic cell) as described herein, comprising delivering a vector as described herein to the cell. The mutations can include the introduction, deletion, or substitution of one or more nucleotides in each target sequence of the cell via a guide RNA or sgRNA. The mutations can include the introduction, deletion, or substitution of 1 to 75 nucleotides in each target sequence of the cell via a guide RNA or sgRNA. The mutations can include the introduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides in each target sequence of the cell via a guide RNA or sgRNA. The mutation may comprise the introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides in each target sequence of the cell via the guide RNA or sgRNA.The mutation may comprise the introduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides in each target sequence of the cell via the guide RNA or sgRNA. The mutation may comprise the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides in each target sequence of the cell via the guide RNA or sgRNA. The mutation may comprise the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400, or 500 nucleotides in each target sequence of the cell via the guide RNA or sgRNA.

[0347] To minimize toxicity and off-target effects, it may be important to control the concentration of the delivered Cas mRNA and guide RNA. The optimal concentration of Cas mRNA and guide RNA can be determined by testing different concentrations in cells or non-human eukaryotic animal models and using deep sequencing to analyze the extent of modification at potential off-target genomic loci. Alternatively, to minimize the level of toxicity and off-target effects, Cas nickase mRNA (e.g., S. pyogenes Cas9 with a D10A mutation) can be delivered together with a pair of guide RNAs targeting the desired site. Guide sequences and techniques for minimizing toxicity and off-target effects can be as described in International Publication No. WO 2014 / 093622 (PCT / US2013 / 074667); or by mutation as described herein.

[0348] Typically, with respect to endogenous CRISPR systems, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). Without wishing to be bound by theory, a tracr sequence that may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence) may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to the guide sequence.

[0349] In certain embodiments, a guide of the present invention comprises a non-natural nucleic acid and / or a non-natural nucleotide and / or a nucleotide analog, and / or a chemical modification. The non-natural nucleic acid can, for example, comprise a mixture of natural and non-natural nucleotides. The non-natural nucleotide and / or nucleotide analog can be modified at the ribose, phosphate, and / or base moiety. In one embodiment of the present invention, the guide nucleic acid comprises ribonucleotides and non-ribonucleotides. In one such embodiment, the guide comprises one or more ribonucleotides and one or more deoxyribonucleotides. In one embodiment of the present invention, the guide comprises one or more non-natural nucleotides or nucleotide analogs, such as a nucleotide having a phosphorothioate bond, a boranophosphate bond, a locked nucleic acid (LNA) nucleotide containing a methylene bridge between the 2' and 4' carbons of the ribose ring, a peptide nucleic acid (PNA), or a bridged nucleic acid (BNA). Other examples of modified nucleotides include 2'-O-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'-fluoro analogs. Further examples of modified nucleotides include the attachment of a chemical moiety at the 2' position, including, but not limited to, peptide, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethylene glycol (TEG). Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ), N 1 -Methylpseudouridine (me 1Ψ), 5-methoxyuridine (5moU), inosine, and 7-methylguanosine. Examples of guide RNA chemical modifications include, but are not limited to, incorporation of 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS), phosphorothioate (PS), S-constrained ethyl (cEt), 2'-O-methyl-3'-thioPACE (MSP), or 2'-O-methyl-3'-phosphonoacetate (MP) at one or more terminal nucleotides. Such chemically modified guides may include increased stability and increased activity compared to unmodified guides, although on-target versus off-target specificity is not predicted. (Hendel,2015,Nat Biotechnol.33(9):985-9,doi:10.1038 / nbt.3290, Published online June 29, 2015;Ragdarm et al.,0215,PNAS,E7110-E7111;Allerson et al. al.,J.Med.Chem.2005,48:901-904;Bramsen et al.,Front.Genet.,2012,3:154;Deng et al.,PNAS,2015,112:11870-11875;Sharma et al.,MedChemComm.,2014,5:1454-1471;Hendel et al. al.,Nat.Biotechnol.(2015)33(9):985-989;Li et al.,Nature Biomedical Engineering, 2017, 1,0066 DOI:10.1038 / s41551-017-0066; Ryan et al., Nucleic Acids Res. (2018) 46(2):792-803). In certain embodiments, the 5' and / or 3' ends of the guide RNA are modified with various functional groups, including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags. (See Kelly et al., 2016, J. Biotech. 233:74-83). In certain embodiments, the guide comprises ribonucleotides in the region that binds to the target DNA and one or more deoxyribonucleotides and / or nucleotide analogs in the region that binds to Cas9, Cpf1, or C2c1.In one embodiment of the present invention, deoxyribonucleotides and / or nucleotide analogs are incorporated into engineered guide structures, including, but not limited to, the 5' and / or 3' ends, stem-loop regions, and seed regions. In certain embodiments, the modifications are absent from the 5'-handle of the stem-loop region. Chemical modifications in the 5'-handle of the stem-loop region of a guide can abolish its function (see Li, et al., Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides of the guide are chemically modified. In some embodiments, 3 to 5 nucleotides at either the 3' or 5' end of the guide are chemically modified. In some embodiments, only a few modifications, e.g., 2'-F modifications, are introduced in the seed region. In some embodiments, 2'-F modifications are introduced at the 3' end of the guide. In certain embodiments, 3 to 5 nucleotides at the 5' and / or 3' end of the guide are chemically modified with 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), 2'-O-methyl-3'-thioPACE (MSP), or 2'-O-methyl-3'-phosphonoacetate (MP). Such modifications can improve genome editing efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9):985-989; Ryan et al., Nucleic Acids Res. (2018) 46(2):792-803). In certain embodiments, all of the phosphodiester bonds of the guide are replaced with phosphorothioate (PS) to improve the level of gene disruption. In certain embodiments, more than five nucleotides at the 5' and / or 3' end of the guide are chemically modified with 2'-O-Me, 2'-F, or S-constrained ethyl (cEt).Such chemically modified guides can mediate enhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS, E7110-E7111). In one embodiment of the present invention, the guide is modified to include a chemical moiety at its 3' and / or 5' end. Such moieties include, but are not limited to, amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), rhodamine, peptide, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethylene glycol (TEG). In certain embodiments, the chemical moiety is conjugated to the guide by a linker such as an alkyl chain. In certain embodiments, the chemical moiety of the modified guide can be used to attach the guide to another molecule, such as DNA, RNA, a protein, or a nanoparticle. Such chemically modified guides can be used to identify or enrich cells commonly edited by the CRISPR system (see Lee et al., eLife, 2017, 6:e25312, DOI:10.7554). In some embodiments, three nucleotides at each of the 3' and 5' ends are chemically modified. In certain embodiments, the modifications include 2'-O-methyl or phosphorothioate analogs. In certain embodiments, 12 nucleotides in the tetraloop and 16 nucleotides in the stem-loop region are substituted with 2'-O-methyl analogs. Such chemical modifications improve in vivo editing and stability (see Finn et al., Cell Reports (2018), 22:2227-2235). In some embodiments, more than 60 or 70 nucleotides of the guide are chemically modified. In some embodiments, the modifications include substitution of nucleotides with 2'-O-methyl or 2'-fluoro nucleotide analogs or phosphorothioate (PS) modifications of phosphodiester linkages. In some embodiments, the chemical modifications include 2'-O-methyl or 2'-fluoro modifications of the guide nucleotides that extend outside the nuclease protein when the CRISPR complex is formed, or PS modifications of 20-30 or more nucleotides at the 3' end of the guide.In certain embodiments, the chemical modifications further include 2'-O-methyl analogs at the 5' end of the guide or 2'-fluoro analogs in the seed and tail regions. Such chemical modifications improve stability against nuclease degradation and maintain or enhance genome editing activity or efficiency, while modifications of all nucleotides may abolish guide function (see Yin et al., Nat. Biotech. (2018), 35(12):1179-1187). Such chemical modifications may be guided by knowledge of the structure of the CRISPR complex, including knowledge of a limited number of nucleases and RNA 2'-OH interactions (see Yin et al., Nat. Biotech. (2018), 35(12):1179-1187). In certain embodiments, one or more guide RNA nucleotides may be replaced with DNA nucleotides. In certain embodiments, up to 2, 4, 6, 8, 10, or 12 RNA nucleotides in the 5'-terminal tail / seed guide region are replaced with DNA nucleotides. In certain embodiments, most of the guide RNA nucleotides at the 3' end are replaced with DNA nucleotides. In certain embodiments, 16 guide RNA nucleotides at the 3' end are replaced with DNA nucleotides. In certain embodiments, 8 guide RNA nucleotides at the 5' end tail / seed region and 16 RNA nucleotides at the 3' end are replaced with DNA nucleotides. In certain embodiments, the guide RNA nucleotides that extend outside the nuclease protein when the CRISPR complex is formed are replaced with DNA nucleotides. This replacement of multiple RNA nucleotides with DNA nucleotides reduces off-target activity but maintains similar on-target activity compared to unmodified guides; however, replacing all RNA nucleotides at the 3' end can disable the function of the guide (see Yin et al., Nat. Chem. Biol. (2018) 14, 311-316).Such modifications can be guided by knowledge of the structure of the CRISPR complex, including knowledge of a limited number of nuclease and RNA 2'-OH interactions (see Yin et al., Nat. Chem. Biol. (2018) 14, 311-316).

[0350] In one embodiment of the present invention, the guide comprises a modified crRNA for Cpf1 having a 5'-handle and a guide segment further comprising a seed region and a 3' end. In some embodiments, the modified guide is selected from the group consisting of Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1); Francisella tularensis subsp. Novicida U112 Cpf1 (FnCpf1); L. bacterium MC2017 Cpf1 (Lb3Cpf1); Butyrivibrio proteoclasticus Cpf1 (BpCpf1); Parcubacteria bacterium GWC2011_GWC2_44_17 Cpf1 (PbCpf1); and Peregrinibacteria bacterium GW2011_GWA_33_10. Cpf1 (PeCpf1); Leptospira inadai Cpf1 (LiCpf1); Smithella sp. SC_K08D17 Cpf1 (SsCpf1); L. bacterium MA2020 Cpf1 (Lb2Cpf1); Porphyromonas crevioricanis Cpf1 (PcCpf1); Porphyromonas macacae Cpf1 (PmCpf1); Candidatus Methanoplasma termitum Cpf1 (CMtCpf1); Eubacterium erigens eligens Cpf1 (EeCpf1); Moraxella bovoculi237 Cpf1 (MbCpf1); Prevotella disiens Cpf1 (PdCpf1); or L. bacterium ND2006 Cpf1 (LbCpf1).

[0351] In some embodiments, the guide modification is a chemical modification, an insertion, a deletion, or a split. In some embodiments, the chemical modification includes, but is not limited to, 2'-O-methyl (M) analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2'-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ), N 1 -Methylpseudouridine (me 1Examples of suitable guide modifications include the incorporation of Ψ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine, 2'-O-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), phosphorothioate (PS), 2'-O-methyl-3'-thioPACE (MSP), or 2'-O-methyl-3'-phosphonoacetate (MP). In certain embodiments, the guide comprises one or more phosphorothioate modifications. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemically modified. In certain embodiments, all nucleotides are chemically modified. In certain embodiments, one or more nucleotides in the seed region are chemically modified. In certain embodiments, one or more nucleotides at the 3' end are chemically modified. In certain embodiments, none of the nucleotides in the 5'-handle are chemically modified. In certain embodiments, the chemical modification in the seed region is a minor modification, such as the incorporation of a 2'-fluoro analog. In certain embodiments, one nucleotide in the seed region is substituted with a 2'-fluoro analog. In certain embodiments, five or ten nucleotides at the 3'-end are chemically modified. Such chemical modifications at the 3'-end of Cpf1 CrRNA improve gene cleavage efficiency (see Li, et al., Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, five nucleotides at the 3'-end are substituted with a 2'-fluoro analog. In certain embodiments, ten nucleotides at the 3'-end are substituted with a 2'-fluoro analog. In certain embodiments, five nucleotides at the 3'-end are substituted with a 2'-O-methyl (M) analog. In certain embodiments, three nucleotides at each of the 3'- and 5'-ends are chemically modified. In certain embodiments, the modifications include 2'-O-methyl or phosphorothioate analogs, hi certain embodiments, 12 nucleotides in the tetraloop and 16 nucleotides in the stem-loop region are substituted with 2'-O-methyl analogs.Such chemical modifications improve in vivo editing and stability (see Finn et al., Cell Reports (2018), 22:2227-2235).

[0352] In some embodiments, the 5'-handle loop of the guide is modified. In some embodiments, the 5'-handle loop of the guide is modified to have a deletion, insertion, split, or chemical modification. In certain embodiments, the loop comprises 3, 4, or 5 nucleotides. In certain embodiments, the loop comprises the sequence UCUU, UUUU, UAUU, or UGUU. In some embodiments, the guide molecule forms a stem-loop with a separate, non-covalently linked sequence, which may be DNA or RNA.

[0353] Synthetically linked guides In one embodiment, the guide comprises a tracr sequence and a tracr mate sequence that are chemically linked or conjugated via a non-phosphodiester bond. In one embodiment, the guide comprises a tracr sequence and a tracr mate sequence that are chemically linked or conjugated via a non-nucleotide loop. In some embodiments, the tracr and tracr mate sequences are linked via a non-phosphodiester covalent linker. Examples of covalent linkers include, but are not limited to, chemical moieties selected from the group consisting of C-C bond-forming groups such as carbamates, ethers, esters, amides, imines, amidines, aminotriazines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, sulphonates, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photolabile bonds, Diels-Alder cycloaddition or ring-closing metathesis pairs, and Michael reaction pairs.

[0354] In some embodiments, tracr and tracr mate sequences are first synthesized using standard phosphoramidite synthesis protocols (Herdewijn, P., ed., Methods in Molecular Biology Col. 288, Oligonucleotide Synthesis: Methods and Applications, Humana Press, New Jersey (2012)). In some embodiments, tracr or tracr mate sequences can be functionalized to contain functional groups suitable for ligation using standard protocols known in the art (Hermanson, G.T., Bioconjugate Techniques, Academic Press (2013)). Examples of functional groups include, but are not limited to, hydroxyl, amine, carboxylic acid, carboxylic acid halide, carboxylic acid activated ester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl, hydrozide, semicarbazide, thiosemicarbazide, thiol, maleimide, haloalkyl, sulfonyl, allyl, propargyl, diene, alkyne, and azide. After functionalization of the tracr and tracr mate sequences, a covalent chemical bond or linkage can be formed between the two oligonucleotides. Examples of chemical bonds include, but are not limited to, those based on C-C bond-forming groups such as carbamates, ethers, esters, amides, imines, amidines, aminotriazines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, sulfones, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photolabile bonds, Diels-Alder cycloaddition or ring-closing metathesis pairs, and Michael reaction pairs.

[0355] In some embodiments, tracr and tracr mate sequences can be chemically synthesized using automated solid-phase oligonucleotide synthesis machines using 2'-acetoxyethyl orthoester (2'-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317:3-18) or 2'-thionocarbamate (2'-TC) chemistry (Dellinger et al., J. Am. Chem. Soc. (2011) 133:11540-11546; Hendel et al., Nat. Biotechnol. (2015) 33:985-989).

[0356] In certain embodiments, tracr and tracr mate sequences can be covalently linked using various bioconjugation reactions, loops, cross-links, and non-nucleotide linkages via modifications of sugars, internucleotide phosphodiester bonds, purine and pyrimidine residues (Sletten et al., Angew. Chem. Int. Ed. (2009) 48:6974-6998; Manoharan, M. Curr. Opin. Chem. Biol. (2004) 8:570-9; Behlke et al., Oligonucleotides (2008) 18:305-19; Watts, et al., Drug. Discov. Today (2008) 13:842-55; Shukla, et al., ChemMedChem (2010) 5:328-49).

[0357] In some embodiments, tracr and tracr mate sequences can be covalently linked using click chemistry. In some embodiments, tracr and tracr mate sequences can be covalently linked using a triazole linker. In some embodiments, tracr and tracr mate sequences are covalently linked using a Huisgen 1,3-dipolar cycloaddition reaction involving an alkyne and an azide to yield a highly stable triazole linker (He et al., ChemBioChem (2015) 17:1809-1812; WO 2016 / 186745). In some embodiments, tracr and tracr mate sequences are covalently linked by ligating a 5'-hexyne tracrRNA and a 3'-azide crRNA. In certain embodiments, either or both of the 5'-hexyne tracrRNA and the 3'-azido crRNA can be protected with a 2'-acetoxyethyl orthoester (2'-ACE) group, which can then be removed using the Dharmacon protocol (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317:3-18).

[0358] In certain embodiments, tracr and tracr mate sequences can be covalently linked via linkers (e.g., non-nucleotide loops) containing moieties such as spacers, bonds, bioconjugates, chromophores, reporter groups, dye-labeled RNA, and non-natural nucleotide analogs. More specifically, suitable spacers for the present invention include, but are not limited to, polyethers (e.g., polyethylene glycol, polyhydric alcohols, polypropylene glycol, or mixtures of ethylene glycol and propylene glycol), polyamine groups (e.g., spermine, spermidine, and their polymeric derivatives), polyesters (e.g., poly(ethyl acrylate)), polyphosphodiesters, alkylenes, and combinations thereof. Suitable linkages include any moiety that can be added to a linker to add additional properties to the linker, such as, but not limited to, a fluorescent label. Suitable bioconjugates include, but are not limited to, peptides, glycosides, lipids, cholesterol, phospholipids, diacylglycerols and dialkylglycerols, fatty acids, hydrocarbons, enzyme substrates, steroids, biotin, digoxigenin, carbohydrates, and polysaccharides. Suitable chromophores, reporter groups, and dye-labeled RNAs include, but are not limited to, fluorescent dyes, such as fluorescein and rhodamine, chemiluminescent, electrochemiluminescent, and bioluminescent marker compounds. Exemplary linker designs for conjugating two RNA components are also described in International Patent Application Publication No. WO 2004 / 015075.

[0359] The linker (e.g., non-nucleotide loop) can be of any length. In some embodiments, the linker has a length equivalent to about 0 to 16 nucleotides. In some embodiments, the linker has a length equivalent to about 0 to 8 nucleotides. In some embodiments, the linker has a length equivalent to about 0 to 4 nucleotides. In some embodiments, the linker has a length equivalent to about 2 nucleotides. Exemplary linker designs are also described in International Patent Application Publication No. WO 2011 / 008730.

[0360] A typical Type II Cas9 sgRNA comprises (from 5' to 3'): a guide sequence, a poly-U tract, a first complementary stretch (the "repeat"), a loop (tetraloop), a second complementary stretch (the "anti-repeat" complementary to the repeat), a stem, and an additional stem-loop and stem and poly-A (often poly-U in RNA) tail (terminator). In preferred embodiments, certain embodiments of the guide structure are retained, and certain embodiments of the guide structure may be modified, for example, by adding, removing, or substituting features, while certain other embodiments of the guide structure are maintained. Preferred locations for engineered sgRNA modifications, including but not limited to, insertions, deletions, and substitutions, include the guide ends and regions of the sgRNA that are exposed when complexed with a CRISPR protein and / or target, e.g., the tetraloop and / or loop 2.

[0361] In certain embodiments, guides of the present invention contain specific binding sites (e.g., aptamers) for adaptor proteins, which may contain one or more functional domains (e.g., via fusion proteins). When such guides form a CRISPR complex (i.e., a CRISPR enzyme bound to the guide and target), the adaptor protein binds and the functional domain associated with the adaptor protein is positioned in a spatial orientation favorable for its attributed function to be effective. For example, if the functional domain is a transcriptional activator (e.g., VP64 or p65), the transcriptional activator will be positioned in a spatial orientation that allows it to affect transcription of the target. Similarly, a transcriptional repressor will be favorably positioned to affect transcription of the target, and a nuclease (e.g., Fok1) will be favorably positioned to cleave or partially cleave the target.

[0362] Those skilled in the art will understand that modifications to the guide that allow for binding of the adaptor + functional domain but do not allow for proper positioning of the adaptor + functional domain (e.g., due to steric hindrance within the three-dimensional structure of the CRISPR complex) are unintended modifications. One or more modified guides can be modified in the tetraloop, stem-loop 1, stem-loop 2, or stem-loop 3, preferably in either the tetraloop or stem-loop 2, and most preferably in both the tetraloop and stem-loop 2, as described herein.

[0363] The repeat:anti-repeat duplex will be evident from the secondary structure of the sgRNA. It will typically consist of a first complementary stretch (from 5' to 3') after the poly-U tract and before the tetraloop; and a second complementary stretch (from 5' to 3') after the tetraloop and before the poly-A tract. The first complementary stretch (the "repeat") is complementary to the second complementary stretch (the "anti-repeat"). Thus, they undergo Watson-Crick base pairing to form the dsRNA duplex when folded against each other. The anti-repeat sequence is therefore complementary to the repeat in terms of AU or CG base pairing and also in terms of the fact that the anti-repeat is in the opposite direction due to the tetraloop.

[0364] In one embodiment of the present invention, the modification of the guide structure comprises substituting bases in stem-loop 2. For example, in one embodiment, the "actt" ("acuu" in RNA) and "aagt" ("aagu" in RNA) bases in stem-loop 2 are substituted with "cgcc" and "gcgg". In one embodiment, the "actt" and "aagt" bases in stem-loop 2 are substituted with a four-nucleotide complementary GC-rich region. In one embodiment, the four-nucleotide complementary GC-rich region is "cgcc" and "gcgg" (both in the 5' to 3' direction). In one embodiment, the four-nucleotide complementary GC-rich region is "gcgg" and "cgcc" (both in the 5' to 3' direction). Other combinations of C and G in the four-nucleotide complementary GC-rich region will be apparent, including CCCC and GGGG.

[0365] In one embodiment, stem loop 2, e.g., "ACTTgtttAAGT" (SEQ ID NO: 36), can be replaced with any "XXXXgtttYYYY" (SEQ ID NO: 37), e.g., where XXXX and YYYY represent any complementary set of nucleotides that will base pair with each other to create the stem.

[0366] In one embodiment, the stem comprises at least about 4 bp comprising complementary X and Y sequences, although stems of more are contemplated, e.g., 5, 6, 7, 8, 9, 10, 11, or 12 or fewer, e.g., 3, 2 base pairs. Thus, for example, X2-12 and Y2-12 (X and Y represent any complementary set of nucleotides) are contemplated. In one embodiment, the stem made of X and Y nucleotides, together with "gttt", forms a perfect hairpin in the overall secondary structure; this can be advantageous, and the amount of base pairs can be any amount that forms a perfect hairpin. In one embodiment, any complementary X:Y base-pairing sequence (e.g., in terms of length) is permissible as long as the secondary structure of the entire sgRNA is preserved. In one embodiment, the stem can be in a form of X:Y base-pairing that does not disrupt the secondary structure of the entire sgRNA, since it has a DR:tracr duplex and a 3-stem-loop. In one embodiment, the "gttt" tetraloop connecting ACTT and AAGT (or any alternative stem made of X:Y base pairs) can be any sequence of the same length (e.g., 4 base pairs) or longer that does not disrupt the overall secondary structure of the sgRNA. In one embodiment, the stem-loop can further extend stem-loop 2, e.g., an MS2 aptamer. In one embodiment, stem-loop 3 "GGCACCGagtCGGTGC" (SEQ ID NO: 38) can similarly take the form "XXXXXXXagtYYYYYYY" (SEQ ID NO: 39), where, for example, X7 and Y7 represent any complementary set of nucleotides that base pair with each other to create the stem. In one embodiment, the stem comprises approximately 7 bp comprising complementary X and Y sequences, although stems of more or fewer base pairs are also contemplated. In one embodiment, the stem made of X and Y nucleotides, together with "agt," will form a perfect hairpin in the overall secondary structure. In one embodiment, any complementary X:Y base pairing sequence is allowed as long as the secondary structure of the entire sgRNA is preserved. In one embodiment, the stem can be in a form of X:Y base pairing that does not disrupt the secondary structure of the entire sgRNA because it has a DR:tracr duplex and three stem loops.In one embodiment, the "agt" sequence of stem-loop 3 can be extended or replaced by an aptamer, e.g., an MS2 aptamer, or a sequence that otherwise generally preserves the structure of stem-loop 3. In an alternative stem-loop 2 and / or 3 embodiment, each X and Y pair can refer to any base pair. In one embodiment, non-Watson-Crick base pairings are contemplated, where such pairings otherwise generally preserve the structure of the stem-loop at that position.

[0367] In one embodiment, the DR:tracrRNA duplex can be substituted with the following sequence (using standard IUPAC nomenclature for nucleotides): gYYYYag(N)NNNNxxxxNNNN(AAN)uuRRRRu (SEQ ID NO: 40), where (N) and (AAN) represent a portion of the bulge in the duplex and "xxxx" represents a linker sequence. The NNNN in the direct repeat can be any sequence as long as it base-pairs with the corresponding NNNN portion of the tracrRNA. In one embodiment, the DR:tracrRNA duplex can be connected by a linker of any length (xxxx...) and any base composition, as long as it does not change the overall s...

Claims

1. 1. A targeting moiety effective for targeting a muscle cell, the targeting moiety comprising one or more n-mer motifs, wherein at least one n-mer motif of the one or more n-mer motifs is selected from the group consisting of X ｍ RGDX ｎ wherein X ｍ and X ｎ are each independently selected from any amino acid, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and m is 1-4, and optionally the n-mer motif is 3-15 amino acids; A composition optionally comprising a cargo, said cargo being linked or otherwise attached to said targeting moiety.

2. 2. The composition of claim 1, wherein the at least one n-mer motif is as in any one of Table 4 (SEQ ID NOS:2-7, 20-21, 41-409), Table 5 (SEQ ID NOS:2, 28, 30-32, 55, 76, 96, 103, 135, 158, 207, 214, 252, 306, 316, 398, 410-768), Figure 14F (SEQ ID NOS:8-12, 14-18), Table 6 (SEQ ID NOS:774-795), Figure 13 (SEQ ID NOS:2-12), Figure 14F (SEQ ID NOS:8-12, 14-18), Figure 19B (2-7, 20-21), Figure 27B (SEQ ID NOS:2-12), Figure 28B (28-32), Figure 28D (SEQ ID NOS:28-30), or any combination thereof.

3. The composition of claim 1 , wherein the targeting moiety comprises a polypeptide, a polynucleotide, a lipid, a polymer, a sugar, or a combination thereof.

4. 2. The composition of claim 1, wherein the targeting moiety comprises a viral protein, and optionally the viral protein is a capsid protein, the viral protein is an adeno-associated virus (AAV) protein, or both.

5. the n-mer motif is located between two amino acids of the viral protein such that the n-mer motif is on the outside of the viral capsid; Optionally, the n-mer motif is inserted between any two consecutive amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide, or at an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide; Optionally, the n-mer motif is inserted between amino acids 588 and 589 in the AAV9 capsid polypeptide, or at a similar position in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide. The composition of claim 4.

6. the composition is an engineered viral particle; Optionally, the engineered viral particle is an engineered AAV viral particle; or Optionally, the AAV viral particle is an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 viral particle. The composition of claim 1.

7. The composition comprises: (a) reduced or eliminated specificity for non-muscle cells, optionally wherein the non-muscle cells are hepatocytes; (b) increased muscle cell capacity; (c) muscle cell specificity; (d) reduced immunogenicity, or (e) any combination thereof 10. The composition of claim 1, wherein

8. the viral capsid protein is an engineered AAV capsid protein that has reduced or eliminated uptake in non-muscle cells compared to a corresponding wild-type AAV capsid polypeptide; Optionally, the wild-type capsid polypeptide is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 capsid polypeptide; Optionally, the engineered AAV capsid protein comprises one or more mutations that reduce or eliminate uptake in non-muscle cells. The composition of claim 6.

9. The one or more mutations are a. 267th place, b. At 269th place, c. At 504th place, d. 505th place, e. At 590th place, f. or any combination thereof, or at one or more corresponding positions in a non-AAV9 capsid polypeptide, Optionally, the non-AAV9 capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide; If desired, the mutation at position 267 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is an A to G or X mutation, where X is any amino acid; the mutation at position 269 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is an S or X to T mutation, where X is any amino acid, or the mutation at position 504 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a G or X to A mutation, where X is any amino acid; the mutation at position 505 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a mutation from P or X to A, where X is any amino acid, or the mutation at position 590 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a mutation from Q or X to A, where X is any amino acid; or the engineered AAV9 capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 267, 269, or both of wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 267 is a G to A mutation and the mutation at position 269 is an S to T mutation; or the engineered AAV capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 590 of the wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 509 is a Q to A mutation; or the engineered AAV9 capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 504, 505, or both of a wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 504 is a G to A mutation and the mutation at position 505 is a P to A mutation; The composition of claim 8.

10. wherein the optional cargo is capable of treating or preventing a muscle disease or disorder; Optionally, the muscle disease or disorder is: a. autoimmune diseases; b. cancer; c. Muscular dystrophy; d. neuromuscular disease; e. sugar or glycogen storage disease; f. repeat expansion disease; g. dominant-negative diseases; h. cardiomyopathy; i. viral diseases; j. Progeroid diseases; or any combination thereof 2. The composition of claim 1, wherein:

11. 2. The composition of claim 1, wherein the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a PPMO, one or more polynucleotides encoding one or more peptides, a CRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein comprising a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other recombinant or therapeutic RNA and / or protein, or any combination thereof.

12. The cargo is (a) capable of inducing exon skipping in a gene, optionally wherein the cargo is capable of inducing exon skipping in the dystrophin gene, or (b) a mini- or micro-dystrophin gene, optionally comprising spectrin-like repeats 1, 2, 3, and 24, and optionally an nNOS domain; The composition of claim 1.

13. (a) the repeat expansion disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD), and optionally the myotonic dystrophy is type 1 or type 2; (b) the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or FSHD; (c) the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-related cardiomyopathy, or Danon disease; (d) the sugar or glycogen storage disease is MPS type III disease or Pompe disease, and optionally the MPS type III disease is MPS type IIIA, IIIB, IIIC, or IIID; or (e) the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia; The composition of claim 10.

14. 1. A vector system comprising a vector, said vector comprising: One or more polynucleotides each encoding all or part of one or more targeting moieties effective to target muscle cells, each targeting moiety comprising one or more n-mer motifs, wherein at least one n-mer motif of the one or more n-mer motifs is selected from the group consisting of X ｍ RGDX ｎ wherein X ｍ and X ｎ are each independently selected from any amino acid, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and m is 1 to 4, and at least one of the one or more polynucleotides encodes at least one of the n-mer motifs; and Optionally, a vector system comprising regulatory elements operably linked to one or more of said polynucleotides.

15. 15. The vector system of claim 14, wherein the at least one n-mer motif is as in any one of Table 4 (SEQ ID NOS:2-7, 20-21, 41-409), Table 5 (SEQ ID NOS:2, 28, 30-32, 55, 76, 96, 103, 135, 158, 207, 214, 252, 306, 316, 398, 410-768), Figure 14F (SEQ ID NOS:8-12, 14-18), Table 6 (SEQ ID NOS:774-795), Figure 13 (SEQ ID NOS:2-12), Figure 14F (SEQ ID NOS:8-12, 14-18), Figure 19B (2-7, 20-21), Figure 27B (SEQ ID NOS:2-12), Figure 28B (28-32), Figure 28D (SEQ ID NOS:28-30), or any combination thereof.

16. The vector system of claim 14 further comprising a cargo.

17. the cargo is a cargo polynucleotide, optionally linked to one or more of the one or more polynucleotides encoding the targeting moiety; Optionally, the cargo polynucleotide is present on the same vector as the one or more polynucleotides encoding the targeting moiety or on a different vector.

17. A vector system according to claim 16.

18. The vector system comprises: (a) a viral particle containing said cargo, if present; (b) a viral polypeptide comprising one or more of said targeting moieties, optionally said viral polypeptide being a capsid polypeptide or an AAV capsid polypeptide, optionally said capsid polypeptide or said AAV capsid polypeptide comprising: engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 viral particles; (c) an AAV viral particle, optionally wherein the AAV viral particle is an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, AAV rh.10 capsid polypeptide, or (d) Any combination of (a) to (c) It is possible to generate A vector system according to claim 14.

19. the one or more polynucleotides encoding one n-mer motif are inserted between two codons corresponding to two amino acids of the viral polypeptide, such that the n-mer motif is on the exterior of the viral capsid; Optionally, the one or more polynucleotides encoding one or more n-mer motifs are inserted between two codons corresponding to any two consecutive amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide, or at analogous positions in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide; Optionally, the one or more polynucleotides encoding one or more n-mer motifs are inserted between the codons corresponding to amino acids 588 and 589 in the AAV9 capsid polynucleotide, or at analogous positions in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide.

19. A vector system according to claim 18.

20. the capsid protein is an engineered AAV capsid protein that has reduced or eliminated uptake in non-muscle cells compared to a corresponding wild-type AAV capsid polypeptide; Optionally, the non-muscle cells are hepatocytes; Optionally, the wild-type capsid polypeptide is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 capsid polypeptide; Optionally, the engineered AAV capsid protein comprises one or more mutations that reduce or eliminate uptake in non-muscle cells.

19. A vector system according to claim 18.

21. The one or more mutations in the AAV9 capsid protein (SEQ ID NO: 1) a. 267th place, b. At 269th place, c. At 504th place, d. 505th place, e. At 590th place, f. or any combination thereof, or at one or more corresponding positions in a non-AAV9 capsid polypeptide, If desired, the non-AAV9 capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide; If desired, the mutation at position 267 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is an A to G or X mutation, where X is any amino acid; the mutation at position 269 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is an S or X to T mutation, where X is any amino acid; the mutation at position 504 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a G or X to A mutation, where X is any amino acid; the mutation at position 505 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a mutation from P or X to A, where X is any amino acid, or the mutation at position 590 in the AAV9 capsid protein (SEQ ID NO: 1) or the corresponding position in a non-AAV9 capsid polypeptide is a mutation from Q or X to A, where X is any amino acid, or the engineered AAV9 capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 267, 269, or both of wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 267 is a G to A mutation and the mutation at position 269 is an S to T mutation; or the engineered AAV9 capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 590 of the wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 509 is a Q to A mutation; or the engineered AAV9 capsid protein is an engineered AAV9 capsid polypeptide comprising a mutation at position 504, 505, or both of a wild-type AAV9 capsid protein (SEQ ID NO: 1), wherein the mutation at position 504 is a G to A mutation and the mutation at position 505 is a P to A mutation; 21. The vector system of claim 20.

22. (a) the vector comprising one or more polynucleotides encoding all or part of the one or more targeting moieties, respectively, does not comprise a splice regulatory element; (b) optionally further comprising a polynucleotide encoding a viral rep protein; (i) the viral rep protein encoding polynucleotide is an AAV rep protein encoding polynucleotide; (ii) the viral rep protein encoding polynucleotide is on the same vector or a different vector as the one or more polynucleotides encoding all or part of one or more targeting moieties, respectively; (iii) the viral rep protein is operably linked to a regulatory element; or (iv) any combination of (i) to (iii) above, or (c) both (a) and (b); A vector system according to claim 14.

23. 15. A polypeptide encoded and / or produced by the vector system of claim 14, optionally wherein the polypeptide is a viral polypeptide or an AAV polypeptide, and optionally wherein the polypeptide has increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof.

24. 15. A particle produced by the vector system of claim 14, optionally comprising a polypeptide produced by or encoded by the vector system, optionally wherein the polypeptide is a viral particle or an AAV polypeptide, optionally wherein the particle is a viral particle, optionally wherein the viral particle is an adeno-associated viral (AAV) particle, a lentiviral particle, or a retroviral particle, optionally wherein the viral particle has muscle-specific tropism, and optionally wherein the polypeptide has increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof.

25. the cargo is capable of treating or preventing a muscle disease or disorder; Optionally, the muscle disease or disorder is: a. autoimmune diseases; b. cancer; c. Muscular dystrophy; d. neuromuscular disease; e. sugar or glycogen storage disease; f. repeat expansion disease; g. dominant-negative diseases; h. cardiomyopathy; i. viral diseases; j. premature aging disorders; or Any combination thereof, 15. The vector system of claim 14, a polypeptide encoded or produced by said vector system, or a particle produced by said vector system.

26. 26. The vector system, polypeptide, or particle of claim 25, wherein the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a PPMO, one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein comprising a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other recombinant or therapeutic RNA and / or protein, or any combination thereof.

27. the cargo is capable of inducing exon skipping in a gene, optionally in the dystrophin gene, or the cargo is a mini- or micro-dystrophin gene, optionally containing spectrin-like repeats 1, 2, 3, and 24, and optionally an nNOS domain; 26. A vector system, polypeptide, or particle according to claim 25.

28. (a) the repeat expansion disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD), optionally wherein the myotonic dystrophy is type 1 or type 2; (b) the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or FSHD; (c) the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-related cardiomyopathy, or Danon disease; (d) the sugar or glycogen storage disease is MPS type III disease or Pompe disease, optionally wherein the MPS type III disease is MPS type IIIA, IIIB, IIIC, or IIID; or (e) the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia; 26. A vector system, polypeptide, or particle according to claim 25.

29. (a) the composition of claim 1; (b) a vector system encoding or capable of producing said composition; (c) a polypeptide encoded or produced by the vector system; (d) a particle comprising or produced by said composition, said vector system, said polypeptide, or any combination thereof; or (e) any combination thereof Cells containing

30. 30. The cell of claim 29, wherein the cell is a prokaryotic or eukaryotic cell.

31. (a) the composition of claim 1; (b) a vector system encoding or capable of producing said composition; (c) a polypeptide encoded or produced by the vector system; (d) a particle comprising said composition, said vector system, said polypeptide, or any combination thereof, or produced by said vector system; (e) a cell comprising (a), (b), (c), (d), or any combination thereof; or (f) any combination thereof; and (g) a pharmaceutically acceptable carrier; 10. A pharmaceutical formulation comprising:

32. (a) the composition of claim 1; (b) a vector system encoding or capable of producing said composition; (c) a polypeptide encoded or produced by the vector system; (d) a particle comprising said composition, said vector system, said polypeptide, or any combination thereof, or produced by said vector system; (e) a cell comprising (a), (b), (c), (d), or any combination thereof; or (f) a pharmaceutical formulation comprising (a), (b), (c), (d), (e), or any combination thereof, and a pharmaceutically acceptable carrier; or (g) any combination thereof to a non-human subject in need thereof.

33. 32. A pharmaceutical formulation according to claim 31 for the treatment of a muscular disease or disorder, comprising: Optionally, the muscle disease or disorder is: (a) autoimmune disease; (b) Cancer; (c) muscular dystrophy; (d) neuromuscular disease; (e) sugar or glycogen storage disease; (f) repeat expansion disease; (g) dominant-negative diseases; (h) cardiomyopathy; (i) viral diseases; (j) a premature aging disorder; or Any combination thereof, Pharmaceutical preparations.

34. (a) the repeat expansion disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD), optionally wherein the myotonic dystrophy is type 1 or type 2; (b) the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or FSHD; (c) the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-related cardiomyopathy, or Danon disease; (d) the sugar or glycogen storage disease is MPS type III disease or Pompe disease, optionally wherein the MPS type III disease is MPS type IIIA, IIIB, IIIC, or IIID; or (e) the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia; 34. The pharmaceutical formulation of claim 33.

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