Gene therapy approach for treating disorders associated with TNNT2

Abstract

The present invention provides expression cassettes and methods that reduce expression of an endogenous TNNT2 gene while providing a non-mutant TNNT2 transgene (tTNNT2), resulting in greatly increased TNNT2 transgene incorporation in the sarcomere than from delivery of the TNNT2 transgene alone.

Description

[0001] GENE THERAPY APPROACH FOR TREATING DISORDERS ASSOCIATED WITH TNNT2

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 638.086, filed April 24. 2024, which is incorporated by reference herein in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to gene therapy approaches.

[0006] BACKGROUND

[0007] Cardiomyopathies represents a collection of heart muscle disfunctions and is the second most common cause of heart disease in subjects. When cardiomyopathy occurs, the normal muscle in the heart can thicken, stiffen, thin out, or fill with substances the body produces that do not belong in the heart muscle. As a result, the heart's ability to pump blood is reduced, which can lead to irregular heartbeats, the backup of blood into the lungs or rest of the body, and heart failure.

[0008] Cardiac troponin T (cTnT) is a protein in cardiac muscle cells that binds tropomyosin to regulate heart contractions and is encoded by the TNNT2 gene. Mutations in the TNNT2 gene can cause familial hypertrophic cardiomyopathy (HCM), a condition characterized by thickening (hypertrophy) of the cardiac muscle, or dialated cardiomyopathy (DCM), a condition characterized by enlargement and thinning of the left ventricle. TNNT2 gene mutations are found in approximately 5 percent of individuals with this condition, with all affected individuals having an increased risk of heart failure and sudden death. Most TNNT2 gene mutations in familial hypertrophic cardiomyopathy or dialated cardiomyopathy change single amino acids in the cTnT protein. The altered protein is incorporated into the troponin complex, resulting in possible dysfunction.

[0009] SUMMARY

[0010] The present invention provides expression cassettes and methods that reduce expression of an endogenous TNNT2 gene while providing a functional TNNT2 transgene (tTNNT2). By the present invention it was discovered that silencing of endogenous TNNT2 expression (wild type and / or mutant) combined with overexpression of tTNNT2 results in increased incorporation of tTNNT2 in the sarcomere than from overexpression of tTNNT2 alone. Remarkably, this synergistic effect may result in greater than double the incorporation of tTNNT2 in the sarcomere, correcting TNNT2 associated cardiomyopathies.

[0011] Accordingly, aspects of the invention provide an expression cassette comprising a first nucleic acid encoding a TNNT2 transgene (tTNNT2) and a second nucleic acid encoding a sequence that reduces expression of endogenous TNNT2. Advantageously, when the first nucleic acid and second nucleic acid are both expressed in a cardiac cell, tTNNT2 protein incorporation in the sarcomere of the cardiac cell is greater than when the first nucleic acid is expressed alone.

[0012] For example, when the first nucleic acid and second nucleic acid are both expressed in the cell, tTNNT2 protein incorporation may about double or greater than double the incorporation level when the first nucleic acid is expressed alone.

[0013] In aspects of the invention, the first nucleic acid and / or second nucleic acid may be operably linked to a promoter. The first nucleic acid and / or second nucleic acid may be operably linked to an enhancer.

[0014] The promoter may comprise a cardiac specific promoter. The promoter may be selected from the group consisting of: TNNT2, Desmin (DES1), and combinations thereof. The expression cassette of claim 1, wherein the first nucleic acid sequence is operably linked to the second nucleic acid sequence.

[0015] In preferred aspects of the invention, the expression cassette comprises a vector, for example a viral vector or lipid nanoparticle (LNP). The vector may transport (e.g. encapsidate or encapsulate) the first nucleic and second nucleic acid. The expression cassette may comprise a first vector transporting the first nucleic acid and a second vector transporting the second nucleic acid.

[0016] The vector may be an adeno-associated viral (AAV) vector, for example an AAV vector modified for increased tropism for cardiac cells.

[0017] The first nucleic acid may encode a codon optimized TNNT2 transgene.

[0018] The second nucleic acid may encode an RNA interefering (RNAi) molecule which interferes with expression of endogenous TNNT2. The RNAi molecule maty be a small interfering RNA (siRNA) or micro RNA (miRNA). The second nucleic acid may encode an antisense oligonucleotide or ribozyme.

[0019] The engoenous TNNT2 may be may be a wild-type (e.g. functional) and / or mutant TNNT2.

[0020] Aspects of the invention also provide methods of treating a disorder associated with mutant TNNT2 by providing to a subject an expression cassette of the invention. For example, aspects of the invention provide a method of treating a disorder associated with mutant TNNT2, the method comprising administering to a subject an expression cassette comprising a first nucleic acid encoding a TNNT2 transgene (tTNNT2) and a second nucleic acid encoding a sequence that reduces expression of endogenous TNNT2. Advantagouesly, expression of the first nucleic acid and second nucleic acid results in greater tTNNT2 protein incorporation than from incorporation of the first nucleic acid alone. For example, expression of the first nucleic acid and second nucleic acid may result in greater than double the tTNNT2 protein incorporation than from expression of the first nucleic acid is alone.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows an electrophoresis assay of 3xFLAG-TNNT2 transgene protein levels in mouse heart.

[0023] FIG. 2 shows a graph of 3xFLAG-TNNT2 transgene expression protein levels in mouse heart.

[0024] FIG. 3 shows a graph of endogenous mouse Tnnt2 mRNA expression in mouse heart.

[0025] DETAILED DESCRIPTION

[0026] The present invention provides expression cassettes and methods that reduce expression of an endogenous TNNT2 gene while providing a non-mutant TNNT2 transgene 0TNNT2), resulting in greatly increased TNNT2 transgene incorporation in the sarcomere than from delivery of the TNNT2 transgene alone.

[0027] Cardiomyopathy

[0028] Cardiomyopathy is a class of disease of heart muscle that adversely impacts the hearts ability to circulate blood through the cardiovascular system. Various types of cardiomyopathies exist, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy. Cardiomyopathy in human populations is a major medical burden and treatment needs are currently unmet, despite cardiomyopathies in human populations being particularly desirable to treat.

[0029] Dilated cardiomyopathy (DCM) is one of the most common types of human cardiomyopathy, occurring mostly in adults 20 to 60. DCM affects the heart's ventricles and atria, the lower and upper chambers of the heart, respectively. Most forms of DCM are acquired forms from a number of causes that include coronary heart disease, heart attack. high blood pressure, diabetes, thyroid disease, viral hepatitis and viral infections that inflame the heart muscle. Alcohol abuse and certain drugs, such as cocaine and amphetamines, as well as at least two drugs used to treat cancer (doxorubicin and daunorubicin), can also lead to DCM. In addition, there are a number of genetic forms of DCM, including, but not limited to the DCM associated with Duchenne and Becker muscular dystrophies. In the case of certain forms of Becker muscular dystrophy, as well as in most cases of Duchenne muscular dystrophy, the cardiomyopathy can ultimately limit the patient’ s survival.

[0030] Hypertrophic cardiomyopathy (HCM) occurs when the walls of the heart muscle become abnormally thick. The increase in wall thickness may increase cardiac complications, as well as block or obstruct blood flowing in the heart.

[0031] Restrictive cardiomyopathy (RCM) is a condition leading to a stiffening of the chambers of the heart over time. While the heart’s ability to contract remains largely unaffected, the cardiac muscle does not fully relax between beats of the heart. This restricts the abi 1 i ty of the ventricles to fdl with blood and causes blood to back up in the circulatory system.

[0032] Heart function is critically dependent upon calcium-dependent signaling. During heart disease, malfunctioning of calcium channels within cardiac cells promotes calcium cycling abnormalities, further inhibiting heart function.

[0033] Point mutations and duplications in the TNNT2 gene cause various types of cardiomyopathies, including hypertrophic cardiomyopathy (HCM). dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM).

[0034] Gene Silencing

[0035] Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation. Gene silencing is used herein interchangeably with gene knockdown and refers to the reduction of expression of a gene. The term “reduced” is used herein to indicate that the target gene expression is lowered by 1-100%. For example, the expression may be reduced by 10, 20, 30, 40. 50. 60, 70, 80, 90, 95, or even 99%. The term “reduced” is used herein to indicate that the target gene expression is lowered by 1-100%. For example, the expression may be reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 99%.

[0036] Any gene silencing method may be used in aspects of the invention.

[0037] For example, antisense oligonucleotides are a gene silencing method in which short nucleic acid fragments bind to complementary target mRNA molecules. These molecules can be composed of single-stranded DNA or RNA and are generally 13-25 nucleotides long. The antisense oligonucleotides can affect gene expression in two ways: by using an RNase IT- dependent mechanism or by using a steric blocking mechanism. RNase H-dependent oligonucleotides cause the target mRNA molecules to be degraded, while steric-blocker oligonucleotides prevent translation of the mRNA molecule. The majority of antisense drugs function through the RNase H-dependent mechanism, in which RNase H hydrolyzes the RNA strand of the DNA / RNA heteroduplex.

[0038] Ribozymes are catalytic RNA molecules used to inhibit gene expression. Ribozymes work by cleaving mRNA molecules. Several types of ribozyme motifs exist, including hammerhead, hairpin, hepatitis delta virus, group I, group II, and RNase P ribozymes. Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs are generally found in viruses or viroid RNAs. These motifs are able to self-cleave a specific phosphodiester bond on an mRNA molecule. Lower eukaryotes and a few bacteria contain group I and group II ribozymes. These motifs can self-splice by cleaving and joining phosphodiester bonds. The RNase P ribozyme, is found in Escherichia coli and is known for its ability to cleave the phosphodiester bonds of several tRNA precursors when joined to a protein cofactor.

[0039] The general catalytic mechanism used by ribozy mes is similar to the mechanism used by protein ribonucleases. These catalytic RNA molecules bind to a specific site and attack the neighboring phosphate in the RNA backbone with their 2' oxygen, which acts as a nucleophile, resulting in the formation of cleaved products with a 2'3'-cyclic phosphate and a 5' hydroxyl terminal end. This catalytic mechanism has been increasingly used by scientists to perform sequence-specific cleavage of target mRNA molecules.

[0040] "RNA interference (RNAi)" is the process of sequence-specific, post-transcriptional gene silencing initiated by short interference RNA (siRNA). RNAi is seen in a number of organisms such as Drosophila, nematodes, fungi and plants, and is believed to be involved in anti-viral defense, modulation of transposon activity, and regulation of gene expression. During RNAi, RNAi molecules induce degradation of target mRNA with consequent sequence-specific inhibition of gene expression.

[0041] A "small interfering” or ‘"short interfering RNA" or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest. A "RNA duplex" refers to the structure formed by the complementary7pairing betw een two regions of a RNA molecule. siRNA is "targeted" to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary7to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23. 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19 - 25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary7in length. In some embodiments the loop is 5. 6, 7, 8, 9, 10. 11. 12 or 13 nucleotides in length. The hairpin structure can also contain 3' or 5' overhang portions. In some embodiments, the overhang is a 3' or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. The “sense” and “antisense” sequences can be used with or without a loop region to form siRNA molecules. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi. for example, double- stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, post- transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetic silencing. For example, siRNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the prc-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression. In another nonlimiting example, modulation of gene expression by siRNA molecules of the invention can result from siRNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or alternately, translational inhibition as is known in the art.

[0042] Embodiments of this disclosure can provide compositions and methods for gene silencing and modulating protein expression using small nucleic acid molecules. Examples of nucleic acid molecules include molecules active in RNA interference (RNAi molecules), short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), or short hairpin RNA (shRNA) molecules, as well as DNA-directed RNAs (ddRNA), Piwi- interacting RNAs (piRNA), or repeat associated siRNAs (rasiRNA). Such molecules are capable of mediating RNA interference against gene expression. In some embodiments, gene silencing can target a specific defective allele. In some embodiments, the gene silenced defective allele can then be replaced by a functional copy. In some embodiments, the functional copy of a gene is codon optimized, such that dissimilarities between a defective copy and a functional copy allow for silencing only of the defective copy. The siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal.

[0043] Cardiac Promoters

[0044] The invention may contain a cardiac specific promoter or another promoter. Promoter regions enable the host cells to replicate the AAV delivered nucleic acid only in those cell ty pes and tissues or organs in which the desired protein should be created. Here, the cardiac specific promoter is included because it is principally desired that the proteins only be translated in myocytes. Specificity of the cell type into which the nucleic acid is delivered and thus the proteins translated is desired because of the adverse effects that may ensue from delivering the nucleic acid and having it translated in cells in which that nucleic acid and thus protein is not needed.

[0045] In some embodiments, the promoter comprises a cardiac specific promoter. In some embodiments, the promoter is TNNT2. In some embodiments, the promoter is MHCK9. In some embodiments, the promoter is MHCK7. In some embodiments, the promoter is CB A (Chicken -Actin). In some embodiments, the promoter is CMV or mini CMV. In some embodiments, the promoter is a Desmin promoter.

[0046] In some embodiments, the promoters described herein are inserted into an AAV protein (e.g., an AAV capsid protein) that has reduced specificity (or no detectable, measurable, or clinically relevant interaction) for one or more non-muscle cell ty pes. Exemplary non-muscle cell types include, but are not limited to, liver, kidney, lung, spleen, central or peripheral nervous system cells, bone, immune, stomach, intestine, eye, skin cells and the like. In some embodiments, the non-muscle cells are liver cells.

[0047] The term “operably linked” refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.

[0048] Vectors

[0049] Gene therapy utilizes the delivery of DNA or RNA into cells through a delivery' system, referred to as a vector. The two major classes of vectors, viral and non-viral. \

[0050] Non-viral vectors depend on physical or chemical methods of delivering genetic material into a cell. This can be either a physical technique or a chemical technique. Physical methods allow researchers to directly deliver genetic material to target cells (for example, using an injection needle). Chemical methods use natural or synthetic materials, for example fat molecules (lipids), polymers, and nanoparticles, or a comination thereof.

[0051] For example, electroporation is a non-viral delivery method being studied to deliver gene therapy in clinical trials. With electroporation, scientists use pulses of electricity' to cause temporary pores to form in a cell membrane. These pores enable gene therapy to be delivered into the cell where it can take effect. Other techniques include the use of a ‘"gene gun,” which uses force), sonoporation (which uses sound), and photoporation (which uses light) to deliver genetic material into a cell.

[0052] The leading non-viral delivery method uses lipid nanoparticles (LNPs). LNPs encapsulate and protect genetic material from degradation before it can reach target cells. LNPs provide the advantage of being quick to manufacture, efficient, and scalable to the size of the material being delivered.

[0053] Viral vectors are built using engineered viral capsids that encapsidate DNA or RNA for delivery’. The most common viral vectors are lentiviral vectors (LVVs) and adenoviral vectors (AAVs). LVVs are a species of retrovirus. Lentiviral vectors have the ability to enter the cell and insert its genetic material into dividing cells (such as stem cells) and non-dividing cells (such as cardiac cells). Lentiviruses also integrate genetic material into the host genome, which alloyvs for continued gene expression.

[0054] Adeno Associated Virus Vector

[0055] AAVs are particularly appropriate viral vectors for delivery of genetic material into mammalian cells. AAVs are not known to cause disease in mammals and cause a very mild immune response. Additionally, AAVs are able to infect cells in multiple stages whether at rest or in a phase of the cell replication cycle. Advantageously, AAV DNA is not regularly inserted into the host’s genome at random sites, reducing the oncogenic properties of this vector.

[0056] AAVs have been engineered to deliver a variety of treatments, especially for genetic disorders caused by single nucleotide polymorphisms (“SNP”). Genetic diseases that have been studied in conjunction with AAV vectors include Cystic fibrosis, hemophilia, arthritis, macular degeneration, muscular dystrophy, Parkinson’s disease, congestive heart failure, and Alzheimer's disease. The AAV can be used as a vector to deliver engineered nucleic acid to a host and utilize the host’s own ribosomes to transcribe that nucleic acid into the desired proteins. See, e.g.. West et al.. Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94: 1351 (1994). AAVs have some deficiency in their replication and / or pathogenicity and thus can be safer that adenoviral vectors. In some embodiments, the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects. In some embodiments, the capacity of the AAV vector, system thereof, and / or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can include one or more engineered capsid polynucleotides described herein.

[0057] AAV s are small, replication-defective, nonenveloped viruses that infect humans and other primate species and have a linear single-stranded DNA genome. Naturally occurring AAV seroty pes exhibit liver tropism. As a result, transfection of non-liver tissue with traditional AAV vectors is impeded by’ the virus's natural liver tropism. Moreover, because the liver acts to break down substances delivered to a subject, transfection of non-liver tissue with unmodified AAV vectors requires higher dosing to provide sufficient viral load to overcome the liver and reach non-liver tissue. More than 30 naturally occurring serotypes of AAV are available. Many natural variants in the AAV capsid exist. AAV serotypes include, but are not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13. AAVs may be engineered using conventional molecular biology techniques, making it possible to optimize these particles, for example, for cell specific delivery, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivers’ to the nucleus. AAV vectors can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery’ method.

[0058] Previous approaches to identify AAV sequences correlated with tropism have relied upon the comparison of highly related extant serotypes with distinct characteristics, random domain swaps between unrelated serotypes, or consideration of higher-order structure, to identify’ motifs that define liver tropism. For example, mapping determinants of AAV tropism have been carried out by comparing highly related seroty pes. One such example is the singleamino acid change (E531K) between AAV1 and AAV6 that improves murine liver transduction in AAV1. See Wu et al. (2006) J. Virol.. 80(22): 11393-7, incorporated by reference herein. Another example is a reciprocal domain swap between AAV2 and AAV8 that alters tropism, but fails to define any robust specific tissue-targeting motifs. See Raupp et al. (201) J. Virol., 86(17):9396-408, incorporated by reference herein. Further, global consideration of structure has only highlighted gross differences between better- or worse- liver-transducers that are more observational than useful in practice. Nam et al (2007) J. Virol., 81(22): 12260-71.

[0059] AAVs exhibiting modified tissue tropism that may be used with the present invention are described in U.S. Patent No. 9,695,220, U.S. Patent No. 9,719,070; U.S. Patent No. 10,119,125; U.S. Patent No. 10,526,584; U.S. Patent Application Publication No. 2018- 0369414; U.S. Patent Application Publication No. 2020-0123504; U.S. Patent Application Publication No. 2020-0318082; PCT International Patent Application Publication No. WO 2015 / 054653; PCT International Patent Application Publication No. WO 2016 / 179496; PCT International Patent Application Publication No. WO 2017 / 100791; and PCT International Patent Application Publication No. WO 2019 / 217911, the entirety of the contents of each of which are incorporated by reference herein.

[0060] The AAV vector or system thereof may include one or more regulatory molecules, such as promoters, enhancers, repressors and the like. In some embodiments, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In some embodiments, the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. In some embodiments, the muscle specific promoter can drive expression of an engineered AAV capsid polynucleotide.

[0061] The 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 a protein shell (an engineered capsid) of the AAV virus particle. The engineered capsid can have a cell-, tissue-, and / or organ-specific tropism.

[0062] The AAV vector or system thereof can be configured to produce AAV particles having 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 combinations thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e g., one can select AAV serotypes 1. 2. 5, 9 or a hybrid capsid AAV-1, AAV-2. AAV-5, AAV-9 or any combination thereof for targeting brain and / or neuronal cells; and one can select AAV-4 for targeting cardiac tissue; and one can select AAV-8 for delivery to the liver. Thus, in some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and / or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV -4 serotype. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.

[0063] It will be appreciated that while the different serotypes can provide some level of cell, tissue, and / or organ specificity, each serotype still is multi-tropic and thus can result in tissuetoxicity if using that seroty pe to target a tissue that the serotype is less efficient in transducing. Thus, in addition to achieving some tissue targeting capacity via selecting an AAV of a particular serotype, it will be appreciated that the tropism of the AAV serotype can be modified by an engineered AAV capsid described herein. As described elsewhere herein, variants of wild-type AAV of any seroty pe can be generated via a method described herein and determined to have a particular cell-specific tropism, which can be the same or different as that of the reference wild-type AAV serotype. In some embodiments, the cell, tissue, and / or specificity of the wild-type serotype can be enhanced (e.g., made more selective or specific for a particular cell type that the serotype is already biased towards). For example, wild- type AAV-9 is biased towards 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 variant of wild-type AAV-9 as described herein, the tropism for nervous cells might be reduced or eliminated and / or the muscle specificity increased such that the nervous specificity appears reduced in comparison, thus enhancing the specificity for muscle as compared to the wild-type AAV-9. As previously mentioned, inclusion of an engineered capsid and / or capsid protein variant 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 variant of AAV-9 can have specificity’ for a tissue other than muscle or brain in humans.

[0064] In some embodiments, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the rAAV2 / 5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed above, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the 2nd plasmid, the 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, in which the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropism displayed 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 the same specificity issues as with the non-hybrid wild-type seroty pes previously discussed.

[0065] Advantages achieved by the wild-type based hybrid AAV systems can be combined with the increased and customizable cell-specificity that can be achieved with the engineered AAV capsids can be combined by generating a hybrid AAV that can include an engineered AAV capsid described elsewhere herein. It will be appreciated that hybrid AAVs can contain an engineered AAV capsid containing a genome with elements from a different serotype than the reference wild-type serotype that the engineered AAV capsid is a variant of. For example, a hybrid AAV can be produced that includes an engineered AAV capsid that is a variant of an AAV-9 seroty pe that is used to package a genome that contains components (e.g., rep elements) from an AAV -2 serotype. As with wild-type based hybrid AAVs previously discussed, the tropism of the resulting AAV particle will be that of the engineered AAV capsid.

[0066] In some embodiments, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In some embodiments, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g., the engineered AAV capsid polynucleotide(s)).

[0067] The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombination and / or cloning methods or techniques can be used to the vector(s) described herein. Suitable recombination and / or cloning techniques and / or methods can include, but not limited to, those described in U.S. Application publication No. US 2004-0171156 Al. Other suitable methods and techniques are described elsewhere herein.

[0068] Construction of recombinant AAV vectors are described in a number of 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 for constructing an AAV or other vector described herein. AAV vectors are discussed elsew here herein. In some embodiments, the vector can have one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site’?). In some 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.

[0069] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a engineered AAV capsid system described herein are as used in the foregoing documents, such as International Patent Application Publications WO WO 2021 / 050974 and WO 2021 / 077000 and PCT International Application No. PCT / US2021 / 042812, the contents of which are incorporated by reference herein.

[0070] Additional AAV vectors are described in International Patent Application Publication WO 2019 / 2071632, the contents of which are incorporated by reference herein.

[0071] Further AAV vectors are described in International Patent Application Publications WO 2020 / 086881 and WO 2020 / 235543, the contents of each of which are incorporated by reference herein.

[0072] Further AAV vectors are described in International Patent Application Publications WO 2005 / 033321; WO 2006 / 110689; WO 2007 / 127264; WO 2008 / 027084; WO 2009 / 073103; WO 2009 / 073104; WO 2009 / 105084; WO 2009 / 134681; WO 2009 / 136977; WO 2010 / 051367; WO 2010 / 138675; WO 2001 / 038187; WO 2012 / 112832; WO 2015 / 054653; WO 2016 / 179496; WO 2017 / 100791; WO 2017 / 019994; WO 2018 / 209154; WO 2019 / 067982; WO 2019 / 195701 ; WO 2019 / 21791 1 ; WO 2020 / 041498; WO 2020 / 210839; U.S. Patent No. 7,906,111; U.S. Patent No. 9,737,618; U.S. Patent No.10,265,417; U.S. Patent No. 10,485,883; U.S. Patent No. 10,695,441; U.S. Patent No. 10,722.598; U.S. Patent No. 8.999,678; U.S. Patent No. 10,301,648; U.S. Patent No. 10,626,415; U.S. Patent No. 9,198,984; U.S. Patent No. 10,155,931; U.S. Patent No. 8,524,219; U.S. PatentNo. 9,206,238; U.S. Patent No. 8,685,387; U.S. Patent No. 9,359,618; U.S. Patent No. 8,231,880; U.S. Patent No. 8,470,310; U.S. Patent No. 9,597,363; U.S. Patent No. 8,940,290; U.S. Patent No. 9,593,346; U.S. Patent No. 10.501,757; U.S. Patent No. 10.786,568; U.S. Patent No. 10.973,928; U.S. Patent No. 10.519.198; U.S. Patent No. 8,846,031; U.S. Patent No. 9,617,561; U.S. Patent No. 9,884,071; U.S. Patent No. 10,406,173; U.S. Patent No. 9,596,220; U.S. Patent No. 9,719,010; U.S. Patent No. 10,117.125; U.S. Patent No. 10,526,584; U.S. Patent No. 10,881,548; U.S. Patent No. 10,738.087; U.S. Patent Publication No. 2011-023353; U.S. Patent Publication No. 2019- 0015527; U.S. Patent Publication No. 2020-155704; U.S. Patent Publication No 2017- 0191079: U.S. Patent Publication No. 2019-0218574; U.S. Patent Publication No. 2020- 0208176: U.S. Patent Publication No. 2020-0325491; U.S. Patent Publication No. 2019- 0055523; U.S. Patent Publication No. 2020-0385689; U.S. Patent Publication No. 2009- 0317417; U.S. Patent Publication No. 2016-0051603; U.S. Patent Publication No. 2016- 00244783; U.S. Patent Publication No. 2017-0183636; U.S. Patent Publication No. 2020- 0263201; U.S. Patent Publication No. 2020-0101099; U.S. Patent Publication No. 2020- 0318082: U.S. Patent Publication No. 2018-0369414; U.S. Patent Publication No. 2019- 0330278; U.S. Patent Publication No. 2020-0231986, the contents of each of which are incorporated by reference herein.

[0073] Additional AAV vectors with modified tropism are described in PCT / US2024 / 010859 and PCT / US2020 / 015200.

[0074] Capsid Protein

[0075] The capsid protein is the shell or coating of the virus that enables its delivery into the host. Without the protein, the nucleic acids would be destroyed by the host without entering into the host cells and beginning transcription and translation. The capsid protein may be in the natural conformation of a naturally occurring AAV, or it may be modified.

[0076] In certain example embodiments, the AAV capsid protein is an engineered AAV capsid protein having reduced or eliminated uptake in a non-muscle cell as compared to a corresponding wild-type AAV capsid polypeptide.

[0077] In some embodiments, the engineered AAV capsid encoding polynucleotide can be included in a polynucleotide that is 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 coupled to a poly adenylation tail. In some embodiments, the poly adenylation tail can be an SV40 poly adenylation tail. In some embodiments, the AAV capsid encoding polynucleotide can be operably coupled 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 muscle), neurons and supporting cells (e.g.. astrocytes, glial cells, Schwann cells, etc ), fat, spleen, liver, kidney, immune cells, spinal fluid cells, synovial fluid cells, skin cells, cartilage, tendons, connective tissue, bone, pancreas, adrenal gland, blood cell, bone marrow cells, placenta, endothelial cells, and combinations thereof. In some embodiments, the promoter can be a constitutive promoter. Suitable tissue specific promoters and constitutive promoters are discussed elsewhere herein and are generally known in the art and can be commercially available. Suitable muscle specific promoters include, but are not limited to CK8, MHCK7, Myoglobin promoter (Mb), Desmin promoter, muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoter.

[0078] Described herein are various embodiments of engineered viral capsids, such as adeno- associated virus (AAV) capsids, that can be engineered to confer cell-specific tropism, such as muscle specific tropism, to an engineered viral particle. Engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids. The engineered capsids can be included in an engineered virus particle (e g., an engineered lentiviral, retroviral, adenoviral, or AAV virus particle), and can confer cell-specific tropism, reduced immunogenicity, or both to the engineered viral particle. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein that can contain a muscle-specific targeting moiety containing or composed of an n-mer motif described elsewhere herein.

[0079] The engineered viral capsid and / or capsid proteins can be encoded by one or more engineered viral capsid polynucleotides. In some embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide. In some embodiments, an engineered viral capsid polynucleotide (e.g.. an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide) can include a 3’ polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

[0080] The engineered viral capsids can be variants of wild-type viral capsid. For example, in some embodiments, the engineered AAV capsids can be variants of wild-ty pe AAV capsids. In some embodiments, the wild-type AAV capsids can be composed of VP1, VP2, VP3 capsid proteins or a combination thereof. In other words, the engineered AAV capsids can include one or more variants of a 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 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 serotype of the wild-ty pe AAV capsid can be AAV-9. The engineered AAV capsids can have a different tropism than that of the reference wild-type AAV capsid. The engineered viral capsid can contain 1-60 engineered capsid proteins. In some embodiments, the engineered viral capsids 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, the engineered viral capsid can contain 0- 59 wild-type viral capsid proteins. In some embodiments, the engineered viral capsid 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 viral capsid proteins.

[0081] In some embodiments, the engineered AAV capsid can contain 1 -60 engineered capsid proteins. In some embodiments, the engineered AAV capsids can contain 1. 2, 3, 4. 5,

[0082] 6, 7, 8, 9, 10, 1 1, 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 can contain 0-59 wild-type AAV capsid proteins. In some embodiments, the engineered AAV capsid 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-ty pe AAV capsid proteins.

[0083] In some embodiments, the engineered viral capsid protein can have an n-mer amino acid motif, where n can be at least 3 amino acids. In some embodiments, n can be 3, 4, 5, 6,

[0084] 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, an engineered AAV capsid can have a 6-mer or 7-mer amino acid motif. In some embodiments, the n-mer amino acid motif can be inserted between two amino acids in the wild-type viral protein (VP) (or capsid protein). In some embodiments, the n-mer motif can be inserted between two amino acids in a variable amino acid region in a viral capsid protein.

[0085] In some embodiments, the n-mer motif can be inserted between two amino acids in a variable amino acid region in an AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (betaB to betal) and an alpha-helix (alphaA) that are conserved in autonomous parvovirus capsids (see e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structural variable regions (VRs) occur in the surface loops that connect the beta-strands, which cluster to produce local variations in the capsid surface. AAVs have 12 variable regions (also referred to as hypervariable regions) (see e.g., Weitzman and Linden. 2011. “Adeno- Associated Virus Biology.” In Snyder, R.O., Moullier, P. (eds.) Totowa. NJ: Humana Press). In some embodiments, one or more / 7-mer motifs can be inserted between two amino acids in one or more of the 12 variable regions in the w ildtype AVV capsid proteins. In some embodiments, the one or more / 7-mer motifs can be 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, the / 7-mer can be inserted between two amino acids in the VR-III of a capsid protein. In some embodiments, the engineered capsid can have an n-mer inserted between any two contiguous amino acids between amino acids 262 and 269, between any two contiguous amino acids between amino acids 327 and 332, between any tw o contiguous amino acids between amino acids 382 and 386, between any two contiguous amino acids between amino acids 452 and 460, between any two contiguous amino acids between amino acids 488 and 505, between any two contiguous amino acids between amino acids 545 and 558, between any two contiguous amino acids between amino acids 581 and 593, between any two contiguous amino acids betw een amino acids 704 and 714 of an AAV9 viral protein. In some embodiments, the engineered capsid can have an / 7-mer inserted between amino acids 588 and 589 of an AAV9 viral protein. In some embodiments, the engineered capsid can have a 7- mer motif inserted between amino acids 588 and 589 of an AAV9 viral protein. In other embodiments, the motif inserted is a 10-mer motif, with replacement of amino acids 586-88 and an insertion before 589. It will be appreciated that / 7-mers can be inserted in analogous positions in AAV viral proteins of other serotypes. In some embodiments as previously- discussed, the n-mer(s) can be inserted between any two contiguous amino acids within the AAV viral protein and in some embodiments the insertion is made in a variable region.

[0086] In some embodiments, the first 1, 2, 3, or 4 amino acids of an n-mer motif can replace 1, 2, 3, or 4 amino acids of a polypeptide into which it is inserted and preceding the insertion site. In some embodiments, the amino acids of the n-mer motif that replace 1 or more amino acids of the polypeptide into which the n-mer motif is inserted come before or immediately before an “RGD"’ in an n-mer motif, referred to as a MyoAAV capsid. Using an AAV as another non-limiting example, one or more of the n-mer motifs can be inserted into e.g., and AAV9 capsid prolylpeptide between amino acids 588 and 589 and the insert can 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 appreciated that this principle can apply in any other insertion context and is not necessarily limited to insertion betw een residues 588 and 589 of an AAV9 capsid or equivalent position in another AAV capsid. It will further be appreciated that in some embodiments, no amino acids in the polypeptide into which the n- mer motif is inserted are replaced by the n-mer motif.

[0087] In some embodiments, the AAV capsids or other viral capsids or compositions can be cardiac-specific. In some embodiments, cardiac-specificity of the engineered AAV or other viral capsid or other composition is conferred by a muscle specific n-mer motif incorporated in the engineered AAV or other viral capsid or other composition described herein. While not 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 the interaction of the viral particle or other composition containing the engineered AAV capsid or other viral capsid or other composition described herein has increased or improved interactions (e.g.. increased affinity) with a cell surface receptor and / or other molecule on the surface of a muscle cell. In some embodiments, the cell surface receptor is 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 selectively expressed on the surface of a muscle cell. In some embodiments, the cell surface receptor or molecule is an integrin or dimer thereof. In some embodiments, the cell surface receptor or molecule is an Vb6 integrin heterodimer.

[0088] In some embodiments, a muscle specific engineered viral particle or other composition described herein containing the muscle-specific capsid, n-mer motif, or musclespecific targeting moiety described herein can have an increased uptake, delivery rate, transduction rate, efficiency, amount, or a combination thereof in a muscle cell as compared to other cells types and / or other virus particles (including but not limited to AAVs) and other compositions that do not contain the muscle-specific n-mer motif of the present invention.

[0089] First-and second-generation muscle specific AAV capsids were developed using a muscle specific promoter and the resulting capsid libraries were screened in mice and nonhuman primates as described elsewhere herein and / or in e.g. WO 2021 / 050974 and WO 2021 / 077000. First and second generation myoAAV capsids were further optimized in mice and non-human primates as previously described to generate enhanced myoAAV capsids.

[0090] Methods of Treatment

[0091] Disclosed herein are methods of treating a disease or condition, or a symptom of the disease or condition, in a subject, comprising administrating of therapeutically effective amount of one or more compositions (e.g., rAAV particle, AAV vector, pharmaceutical composition) disclosed herein to the subject. In some embodiments, the composition is a rAAV capsid protein described herein. In some embodiments, the composition is an isolated and purified rAAV capsid protein described herein. In some embodiments, the rAAV particle encapsidates an AAV vector comprising a transgene (e.g., therapeutic nucleic acid). In some embodiments, the composition is a rAAV capsid protein described herein conjugated with a therapeutic agent disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising the rAAV particle and a pharmaceutically acceptable carrier. In some embodiments, the one or more compositions are administered to the subject alone (e.g., stand-alone therapy). In some embodiments, the composition is a first-line therapy for the disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line therapy, for the disease or condition.

[0092] Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid (e.g., a transgene, therapeutic nucleic acid). Upon delivery to a host in vivo environment, a rAAV will (1) bind or attach to cellular surface receptors on the target cell. (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid , (5) convert of the heterologous nucleic acid from single-stranded to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe of the episomal heterologous nucleic acid in the nucleus of the host cell (“transduction’'). rAAVs engineered to have an increased transduction enrichment (transcription of the episomal heterologous nucleic acid in the host cell) are desirable for gene therapy applications.

[0093] Aspects disclosed herein provide methods of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the rAAV of the present disclosure, or the pharmaceutical formulation of the present disclosure, wherein the gene product is a therapeutic gene product. In some embodiments, the administering is by intracranial, intraventricular, intracerebroventricular, intravenous, intraarterial, intranasal, intrathecal, intracistemal, or subcutaneous.

[0094] Provided here, are methods of treating a disease or a condition associated with an aberrant expression or activity of a target gene or gene expression product thereof, the method comprising modulating the expression or the activity of a target gene or gene expression product in a subject by administering a rAAV encapsidating a heterologous nucleic acid of the present disclosure. In some instances, the expression or the activity of the target gene or gene expression product is decreased, relative to that in a normal (non- diseased) individual; and administering the rAAV to the subject is sufficient to increase the expression of the activity’ of the target gene or gene expression product. In some instances, the expression or the activity7of the gene or gene expression product is increased, relative to that in a normal individual; and administering the rAAV to the subject is sufficient to decrease the expression or the activity of the target gene or gene expression product.

[0095] Also provided are methods of preventing a disease or condition disclosed herein in a subject comprising administering to the subject a therapeutically effective amount of an rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product described herein. The rAAV vector may be encapsidated in the modified capsid protein or rAAV viral particle described herein. In some instances, the therapeutic gene expression product is effective to modulate the activity or expression of a target gene or gene expression product.

[0096] The compositions of the present disclosure are particularly useful for the treatment of the diseases or conditions described herein because they specifically or more efficiently target the in vivo environment and deliver a therapeutic nucleic acid engineered to modulate the activity or the expression of a target gene expression product involved with the pathogenesis or pathology of the disease or condition.

[0097] Provided herein are methods of treating a disease or a condition, or a symptom of the disease or condition, in a subject, comprising: (a) diagnosing a subject with a disease or a condition affecting a target in vivo environment; and (b) treating the disease or the condition by administering to the subject a therapeutically7effective amount of a composition disclosed herein (e.g., rAAV particle, AAV vector, pharmaceutical composition), wherein the composition is engineered with an increased specificity for the target in vivo environment.

[0098] Disclosed herein are methods of treating a disease or a condition, or a symptom of the disease or the condition, afflicting a target in a subject comprising: (a) administering to the subject a composition (e.g., rAAV particle, AAV vector, pharmaceutical composition); and (b) expressing the therapeutic nucleic acid into a target in vivo environment in the subject with an increased transduction enrichment.

[0099] Also provided are methods of modulating a target gene expression product, the methods comprising administering to a subject in need thereof a composition (e.g., rAAV particle, AAV vector, pharmaceutical composition) disclosed herein. For example, methods provided herein comprise administering to a subject a rAAV with a rAAV capsid protein encapsidating a viral vector comprising a heterologous nucleic acid that modulates the expression or the activity7of the target gene expression product. Pharmaceutical Composition

[0100] Some embodiments of the invention may include any acceptable form of providing the AAV vector to a subject. For example, the AAV vector may be provided to the subject in the form of a composition or formulation comprising the AAV vector. The expression vector of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the subject. The compositions, polynucleotides, polypeptides, particles, cells, vector systems and combinations thereof described herein can be contained in a formulation, such as a pharmaceutical formulation. In some embodiments, the formulations can be used to generate polypeptides and other particles that include one or more muscle-specific targeting moieties described herein. In some embodiments, the formulations can be delivered to a subject in need thereof. In some embodiments, component(s) of the engineered AAV capsid system, engineered cells, engineered AAV capsid particles, and / or combinations thereof described herein can be included in a formulation that can be delivered to a subject or a cell. In some embodiments, the formulation is a pharmaceutical formulation. One or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be provided to a subject in need thereof or a cell alone or as an active ingredient, such as in a pharmaceutical formulation. As such, also described herein are pharmaceutical formulations containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein. In some embodiments, the pharmaceutical formulation can contain an effective amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The pharmaceutical formulations described herein can be administered to a subject in need thereof or a cell.

[0101] In some embodiments, the amount of the one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery' particles, and combinations thereof described herein contained in the pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg based upon the body weight of the subject in need thereof or average body weight of the specific patient population to which the pharmaceutical formulation can be administered. The amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein in the pharmaceutical formulation can range from about 1 pg to about 10 g, from about 10 nL to about 10 ml. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1 x 102, I x IO3, I x 104, 1 x 105, I x 106, 1 x 107, 1 x IO8, 1 x 109, 1 x IO10or more cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x IO10or more cells per nL, pL, mL, or L.

[0102] In embodiments, were engineered AAV capsid particles are included in the formulation, the formulation can contain 1 to 1 x 102, 1 x 103. 1 x 104, 1 x 105, 1 x 106. 1 x 107, 1 x 108, 1 x 109. 1 x IO10, 1 x 1011, 1 x 1012. 1 x 1013. 1 x 1014, 1 x 1015, 1 x 1016. 1 x 1017, 1 x 1018, 1 x 1019, or 1 x IO20transducing units (TU) / mL of the engineered AAV capsid particles. In some embodiments, the formulation can be 0. 1 to 100 mL in volume and can contain 1 to 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x IO10, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014. 1 x 1015, 1 x 1016, 1 x 1017. 1 x 1018, 1 x 1019, or 1 x IO20transducing units (TU) / mL of the engineered AAV capsid particles.

[0103] Pharmaceutically Acceptable Carriers and Auxiliary Ingredients and Agents

[0104] In embodiments, the pharmaceutical formulation containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein can further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.

[0105] The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and / or aromatic substances, and the like which do not deleteriously react with the active composition.

[0106] In some embodiments, the pharmaceutical formulations described herein may be in a dosage form. The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavemous, intrathecal, intravitreaL intracerebral, gingival, subgingival, intracerebroventricular, and intradermal. Such formulations may be prepared by any method known in the art.

[0107] Dosage forms adapted for oral administration can be discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or nonaqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as foam, spray, or liquid solution. In some embodiments, the oral dosage form can contain about 1 ng to 1000 g of a pharmaceutical formulation containing a therapeutically effective amount or an appropriate fraction thereof of the targeted effector fusion protein and / or complex thereof or composition containing the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The oral dosage form can be administered to a subject in need thereof.

[0108] Where appropriate, the dosage forms described herein can be microencapsulated. The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be the ingredient whose release is delayed. In other embodiments, the release of an optionally included auxiliary ingredient is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets," eds. Liberman et. al. (New York, Marcel Dekker, Inc.. 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more.

[0109] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0110] Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and / or pH dependent polymers, with or without water insoluble / water soluble non-polymeric excipient, to produce the desired release profile. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, "ingredient as is" formulated as, but not limited to, suspension form or as a sprinkle dosage form.

[0111] Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treatments of the eye or other external tissues, for example the mouth or the skin, the pharmaceutical formulations are applied as a topical ointment or cream. When formulated in an ointment, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be formulated with a paraffinic or water- miscible ointment base. In some embodiments, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.

[0112] Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is contained in a dosage form adapted for inhalation is in a particle-size-reduced form that is obtained or obtainable by micronization. In some embodiments, the particle size of the size reduced (e.g., micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions / suspensions of an active ingredient (e.g., the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and / or auxiliary active agent), which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.

[0113] In some embodiments, the dosage forms can be aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation can contain a solution or fine suspension of the one or more of the polypeptides, polynucleotides. vectors, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multidose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal, or an aerosol dispenser fitted with a metering valve (e.g., metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.

[0114] Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosol formulation can also contain a solution or a suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. In further embodiments, the aerosol formulation can also contain co-solvents and / or modifiers incorporated to improve, for example, the stability and / or taste and / or fine particle mass characteristics (amount and / or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1 , 2, or 3 doses are delivered each time.

[0115] For some dosage forms suitable and / or adapted for inhaled administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, an auxiliary active ingredient, and / or pharmaceutically acceptable salt thereof, such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and / or metals salts of stearic acid, such as magnesium or calcium stearate.

[0116] In some embodiments, the aerosol dosage forms can be arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.

[0117] Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas. Dosage forms adapted for parenteral administration and / or adapted for any type of injection (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavemous, gingival, subgingival, intrathecal, intravitreal, intracerebral, and intracerebroventricular) can include aqueous and / or non-aqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single- unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets.

[0118] Dosage forms adapted for ocular administration can include aqueous and / or nonaqueous sterile solutions that can optionally be adapted for injection, and which can optionally contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the eye or fluid contained therein or around the eye of the subject, and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents.

[0119] For some embodiments, the dosage form contains a predetermined amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein per unit dose. In some embodiments, the predetermined amount of the Such unit doses may therefore be administered once or more than once a day. Such pharmaceutical formulations may be prepared by any of the methods well know n in the art.

[0120] INCORPORATION BY REFERENCE

[0121] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0122] EQUIVALENTS

[0123] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

[0124] EXAMPLES

[0125] In order that the invention described may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods and compositions provided herein and are not to be construed in any way as limiting their scope.

[0126] Example 1: Endogenous TNNT2 and TNNT2 transgene expression in mice

[0127] C57BL / 6 mice were divided into four cohorts and injected with: (i) a vehicle, (ii) MyoAAV-3xFLAG mTNNT2 (mouse Tnnt2 (iii) MyoAAV-3xFLAG mTNNT2 + MyoAAV-scrambled miRNA, (iii) or MyoAAV-3xFLAG-mTNNT2 + MyoAAV- endogenous mTNNT2 miRNA

[0128] Tissues were harvested from mice two weeks after injection. Incorporation of the mTNNT2 transgene into the sarcomere was quantified by capillary electrophoresis. Endogenous mouse Tnnl2 transcript was quantified by qPCR.

[0129] FIG. 1 shows an electrophoresis assay of 3xFLAG-TNNT2 transgene protein levels in mouse heart.

[0130] FIG. 2 shows a graph of 3xFLAG-TNNT2 transgene protein levels in mouse heart.

[0131] FIG. 3 shows a graph of endogenous mouse Tmt2 mRNA expression in mice. Overexpression of mouse TNNT2 alone or with scrambled miRNA (scrambled knockdown) resulted in 25-36% 3xFLAG-TNNT2 transgene protein incorporation in the sarcomere with no reduction of endogenous TNNT2 expression.

[0132] Overexpression of mouse TNNT2 together with targeted knockdown of endogenous mouse Tnnt2 resulted in an increase of 3xFLAG-TNNT2 transgene incorporation in the sarcomere from 25-36% to 63%. Knocking down endogenous mouse Tnnt2 and overexpressing the 3xFLAG-TNNT2 transgene at the same time greatly increased incorporation of the 3xFLAG-TNNT2 transgene protein into the sarcomere.

Claims

CLAIMSWhat Is Claimed Is:

1. An expression cassette comprising: a first nucleic acid encoding a TNNT2 transgene (tTNNT2); and a second nucleic acid encoding a sequence that reduces expression of endogenous TNNT2, wherein when the first nucleic acid and second nucleic acid are both expressed in a cardiac cell, tTNNT2 protein incorporation in the cardiac cell sarcomere is greater than when the first nucleic acid is expressed alone.

2. The expression cassette of claim 1 , wherein when the first nucleic acid and second nucleic acid are both expressed in cell, tTNNT2 protein incorporation is greater than double the incorporation level when the first nucleic acid is expressed alone.

3. The expression cassette of claim 1, wherein the first nucleic acid and / or second nucleic acid is operably linked to a promoter.

4. The expression cassette of claim 3, wherein the first nucleic acid and / or second nucleic acid is operably linked to an enhancer.

5. The expression cassette of claim 3, wherein the promoter comprises a cardiac specific promoter.

6. The expression cassette of claim 5, wherein the promoter is selected from the group consisting of: TNNT2, Desmin (DES1), and combinations thereof.

7. The expression cassette of claim 1, wherein the first nucleic acid sequence is operably linked to the second nucleic acid sequence.

8. The expression cassette of claim 1, wherein the expression set comprises a vector selected from a viral vector or lipid nanoparticle (LNP).

9. The expression cassete of claim 8, wherein the vector is a viral vector encapsidating the first nucleic acid and second nucleic acid.

10. The expression cassete of claim 9, wherein the vector is an adeno-associated viral (AAV) vector.

11. The expression cassete of claim 10. wherein the AAV vector is modified for increased tropism for cardiac cells.

12. The expression cassete of claim 1, wherein the first nucleic acid encodes a codon optimized TNNT2 transgene.

13. The expression cassete of claim 1, wherein the second nucleic acid encodes an RNA interefering (RNAi) molecule which interferes with expression of endogenous wild ty pe or mutant TNNT2.

14. The expression cassete of claim 13, wherein the RNAi molecule is small interfering RNA (siRNA) or micro RNA (miRNA).

15. The expression cassete of claim 1, wherein the second nucleic acid encodes an antisense oligonucleotide or ribozyme.

16. A method of treating a disorder associated with mutant TNNT2, the method comprising administering to a subject an expression cassete comprising: a first nucleic acid encoding a TNNT2 transgene (tTNNT2); and a second nucleic acid encoding a sequence that reduces expression of endogenous TNNT2, wherein expression of the first nucleic acid and second nucleic acid results in greater tTNNT2 protein incorporation than from expression of the first nucleic acid alone.

17. The method of claim 16, wherein expression of the first nucleic acid and second nucleic acid results in greater than double the tTNNT2 protein incorporation than from expression of the first nucleic acid alone.

18. The method of claim 16, wherein the first nucleic acid and / or second nucleic acid is operably linked to a promoter.

19. The method of claim 18, wherein the first nucleic acid and / or second nucleic acid is operably linked to an enhancer.

20. The method of claim 18. wherein the promoter comprises a cardiac specific promoter.

21. The method of claim 20, wherein the promoter is selected from the group consisting of: TNNT2, Desmin (DES1), and combinations thereof.

22. The method of claim 16, wherein the first nucleic acid sequence is operably linked to the second nucleic acid sequence.

23. The method of claim 16, wherein the expression set comprises a vector selected from a viral vector or lipid nanoparticle (LNP).

24. The method of claim 23, wherein the vector is a viral vector encapsidating the first nucleic acid and second nucleic acid.

25. The method of claim 24, wherein the vector is an adeno-associated viral (AAV) vector.

26. The expression cassette of claim 25, wherein the AAV vector is modified for increased tropism for cardiac cells.

27. The method of claim 16, wherein the first nucleic acid encodes a codon optimized TNNT2 transgene.

28. The method of claim 16, wherein the second nucleic acid encodes an RNA interefering (RNAi) molecule which interferes with expression of mutant TNNT2.

29. The method of claim 28, wherein the RNAi molecule is small interfering RNA (siRNA) or micro RNA (miRNA).

30. The expression cassette of claim 16, wherein the second nucleic acid encodes an antisense oligonucleotide or ribozyme.

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