Composition for treating XLMTM

Abstract

The present invention provides a novel muscle affinity viral vector that achieves MTM1 expression in skeletal muscle. Advantageously, by increasing the expression of MTM1 in skeletal muscle, the vector of the present invention enables administration of a dosage that substantially reduces the exposure of non-target tissues to the composition containing the vector. With more effective dosing, a method of treatment is made possible with improved clinical efficacy (improvement in muscle strength and reduction in the need for mechanical ventilation) and safety, particularly with a reduced risk of hepatotoxicity in this population with a large unmet medical need.

Description

Technical Field

[0001] Field of Disclosure The present disclosure relates to methods for treating X-linked myotubular myopathy (XLMTM).

[0002] Sequence Listing This application includes a sequence listing submitted in electronic form as an ASCII.txt file named KATE-014-00US-Sequence-Listing.txt, created on May 24, 2022, and having a size of 1642.5 KB. The contents of the sequence listing are hereby incorporated by reference in their entirety.

Background Art

[0003] Background X-linked myotubular myopathy (XLMTM) is a rare and severe congenital neuromuscular disease that occurs in approximately 1 in 50,000 male births. XLMTM is caused by pathogenic variants in the myotubularin (MTM1) gene, which encodes the ubiquitously expressed lipid phosphatase protein myotubularin that regulates intracellular membrane trafficking and vesicular transport, and whose function is required for normal development, maturation, and maintenance of skeletal muscle. Reduction in functional myotubularin is associated with profound skeletal muscle weakness that causes the most prominent clinical symptoms of this disease.

[0004] Almost all male infants with XLMTM present at birth with abnormal Apgar scores, severe hypotonia and weakness, and respiratory difficulties, and approximately 90% require mechanical ventilation assistance. Affected male infants have a dramatically shortened lifespan and typically die at a median age of approximately 18 months due to respiratory failure and its complications.

[0005] As highlighted in a recently published chart review, the quality of life is also low for the majority of affected male infants. Patients spend approximately one-third to one-half of the first year of life in the hospital, and readmissions for pneumonia and other respiratory symptoms are common. Even with aggressive supportive therapy to prolong life, male infants with XLMTM reach motor milestones late, if at all, and rarely achieve independent voluntary movement. Most require mechanical ventilation support for life, and about half require 24-hour mechanical ventilation per day. Surgical procedures are also common, and tracheostomy is often required for patients who are dependent on long-term mechanical ventilation, and almost all patients require gastrostomy due to debilitation that impairs their ability to eat.

[0006] Advances in diagnostic techniques, particularly genetic testing, have shortened the time to diagnosis of XLMTM, and it is now typically diagnosed within 3 to 4 months after birth. Although efforts are underway to develop treatment methods, there are currently no pharmaceuticals approved for the treatment of XLMTM. Supportive therapies such as those described above can extend survival, but do not alter the course of the disease.

[0007] Investigational treatment strategies include AT132 (resamiligen bilparvovec), which is an intravenous gene therapy consisting of an AAV8 capsid that delivers human MTM1 along with the desmin promoter. To date, selected data have been published on 23 participants who received AT132 in a Phase 1 / 2 clinical trial (ASPIRO, NCT03199469). This approach has shown a substantial improvement in clinical outcomes, with many participants experiencing improved motor function as evaluated by CHOP-INTEND, a significant reduction in the need for ventilatory support, and some participants being completely liberated from mechanical ventilation.

[0008] Unfortunately, AT132 was also associated with serious safety findings, particularly in four participants where severe intrahepatic cholestasis led to liver failure and death. Hepatotoxicity has also been reported in other gene therapies and typically involves a marked increase in transaminases, which can recover with supportive care and sometimes in combination with steroids. In contrast, hepatotoxicity in the AT132 program is characterized by cholestasis, which can be severe and progressive in some cases, does not respond to immunosuppression according to reports, and is not prevented by the addition of prophylactic ursodeoxycholic acid.

[0009] As a result, there are still no approved medicines for the treatment of XLMTM, and the population of people affected by this condition remains with large unmet medical needs. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0010] Abstract The present invention provides a novel muscle - affinity viral vector that achieves MTM1 expression in skeletal muscle. Advantageously, by increasing MTM1 expression in skeletal muscle, the vectors of the present invention enable administration of a dose that substantially reduces exposure of non - target tissues to the composition containing the vector. With more effective dosing, XLMTM can be treated with improved clinical efficacy (improvement in muscle strength and reduction in the need for mechanical ventilation) and safety, particularly with a reduced risk of hepatotoxicity in this population with large unmet medical needs.

[0011] The method of the present invention provides an AAV vector comprising a capsid protein that includes at least one modification that results in preferential targeting of the AAV vector to muscle tissue. The vector further comprises a nucleic acid encoding the full - length MTM1 protein.

[0012] The capsid protein may further include at least one modification that results in a decrease in the liver affinity of the AAV vector.

[0013] The AAV can be any known AAV, such as AAV9. The capsid protein can include at least one modification that is an insertion between any two consecutive amino acids among amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714 in the AAV9 capsid polypeptide, or insertions at similar positions in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptides. For example, the capsid protein can include at least one modification that is a replacement of amino acids 586-588 and an insertion between amino acids 588 and 589 in the AAV9 capsid polypeptide, or replacements and insertions at similar positions in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptides. The capsid protein may include at least one modification that is a replacement of amino acids 586-588 and an insertion between amino acids 588 and 589 in the AAV9 capsid polypeptide, and the insertion is selected from the sequences of Tables 1-4 provided in detail below.

[0014] The vector can include vp1, vp2, and vp3 capsid proteins. The amino acid sequence of the vp1 capsid protein may be selected from the sequences of Table 5, the amino acid sequence of the vp2 capsid protein may be selected from the sequences of Table 6, and / or the amino acid sequence of the vp3 capsid protein is selected from the sequences of Table 7, each of which is provided in detail below.

[0015] The nucleic acid encoding the full-length MTM1 protein can be operably linked to a muscle-specific promoter. The muscle-specific promoter can be any known muscle-specific promoter. For example, the muscle-specific promoter is the MHCK7 promoter.

[0016] The nucleic acid encoding the full-length MTM1 protein may contain an exon cassette that has undergone alternative splicing downstream of a muscle-specific promoter. The exon cassette that has undergone alternative splicing may contain an ATG start codon at the 3' end of the cassette. The exon cassette that has undergone alternative splicing may contain a skeletal muscle-specific exon. Advantageously, the exon cassette that has undergone alternative splicing may promote skeletal muscle expression of the nucleic acid. As a result, both the AAV capsid and the nucleic acid encoding MTM1 delivered by the capsid result in increased skeletal muscle expression.

[0017] Aspects of the invention provide a method of treating X-linked myotubular myopathy (XLMTM). The method includes administering to a subject afflicted with XLMTM a composition comprising a capsid protein comprising at least one modification that results in preferential targeting of an adeno-associated virus (AAV) vector to muscle tissue, and an AAV vector comprising a nucleic acid encoding a full-length MTM1 protein.

[0018] The capsid protein may further comprise at least one modification that results in a decrease in liver tropism of the AAV vector.

[0019] The AAV can be any known AAV, such as AAV9. The capsid protein can include at least one modification that is an insertion between any two consecutive amino acids among amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714 in the AAV9 capsid polypeptide or an insertion at a similar position in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptides. For example, the capsid protein can include at least one modification that is a replacement of amino acids 586-588 and an insertion between amino acids 588 and 589 in the AAV9 capsid polypeptide or a replacement and an insertion at a similar position in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptides. The capsid protein may include at least one modification that is a replacement of amino acids 586-588 and an insertion between amino acids 588 and 589 in the AAV9 capsid polypeptide, and the insertion is selected from the sequences of Tables 1-4 provided in detail below.

[0020] The vector can include vp1, vp2, and vp3 capsid proteins. The amino acid sequence of the vp1 capsid protein may be selected from the sequences of Table 5, the amino acid sequence of the vp2 capsid protein may be selected from the sequences of Table 6, and / or the amino acid sequence of the vp3 capsid protein is selected from the sequences of Table 7, each of which is provided in detail below.

[0021] The nucleic acid encoding the full-length MTM1 protein can be operably linked to a muscle-specific promoter. The muscle-specific promoter can be any known muscle-specific promoter. For example, the muscle-specific promoter is the MHCK7 promoter.

[0022] The nucleic acid encoding the full-length MTM1 protein may contain an exon cassette that has undergone alternative splicing downstream of a muscle-specific promoter. The exon cassette that has undergone alternative splicing may contain an ATG start codon at the 3' end of the cassette. The exon cassette that has undergone alternative splicing may contain a skeletal muscle-specific exon. Advantageously, the exon cassette that has undergone alternative splicing may promote skeletal muscle expression of the nucleic acid. As a result, both the AAV capsid and the nucleic acid encoding MTM1 delivered by the capsid result in increased skeletal muscle expression.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 3-1

Table 3-2

Table 3-3

Table 4-1

Table 4-2

Table 4-3

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Table 5-36

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Brief Description of the Drawings

[0023]

Fig. 1A-B

Fig. 1C

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Modes for Carrying Out the Invention

[0024] Detailed Description The present invention provides a novel muscle - affinity viral vector that achieves MTM1 expression in skeletal muscle, with a decrease in vector exposure in liver tissue. Advantageously, by increasing the expression of MTM1 in skeletal muscle, the vectors of the present invention enable the administration of a dose that substantially reduces the exposure of non - target tissues to the composition containing the vector.

[0025] XLMTM and AT132 XLMTM is caused by a pathogenic variant in the myotubularin (MTM1) gene, which encodes the ubiquitously expressed lipid phosphatase protein myotubularin that regulates intracellular membrane trafficking and vesicular transport, and whose function is required for normal development, maturation, and maintenance of skeletal muscle. A decrease in functional myotubularin is associated with severe skeletal muscle weakness that causes the most prominent clinical symptoms of this disease.

[0026] MTM1 is a member of a large evolutionarily conserved family of myotubularin phosphatases. It is a ubiquitously expressed lipid phosphatase that dephosphorylates the D3 phosphate of the inositol ring of two phosphoinositides, phosphatidylinositol 3 - phosphate (PtdIns3P) and phosphatidylinositol 3,5 - bisphosphate (PtdIns(3,5)P2). MTM1 regulates numerous cellular processes, including vesicular sorting through the endosomal compartment, excitation - contraction coupling, and T - tubule organization in muscle. Loss - of - function mutations in the MTM1 gene cause defects in these processes, which are thought to underlie the severe motor dysfunction and histological defects in muscles affected by XLMTM.

[0027] Loss of MTM1 gene function in mice and dogs is associated with functional and histological defects similar to those in human children with XLMTM, including decreased lifespan, impaired motor function, muscle atrophy, reduced contractility, and histological defects related to organelle organization including central nuclei, abnormal mitochondrial localization, and T-tubule disorganization. The human MTM1 coding sequence is set forth in SEQ ID NO: 3. Restoration of functional myotubularin by AAV8-mediated gene therapy to express full-length mouse MTM1 under the control of the desmin promoter has been shown to improve histological defects in lifespan, weight gain, muscle contractility, motor function, and organelle localization abnormalities when administered by intramuscular or intravenous (IV) injection into Mtm1 knockout (KO) mice.

[0028] Without being restricted by the mechanism of action, XLMTM is thought to produce abnormal hepatic substrates that are not themselves critical but are more vulnerable to further injury. Hepatic abnormalities, including cholestasis, are part of the natural history of XLMTM. Hepatic symptoms are less prominent than the weakness of skeletal and respiratory muscles, which are the most prominent and very frequently lethal clinical manifestations, but hepatic symptoms can be more clinically important and may be more recognized following the appearance of hepatotoxicity in the AT132 program. Cholestatic liver failure has not been reported as part of the natural history of this disease, but there are case reports of substantial increases in bilirubin in the presence of stress factors such as respiratory infections, consistent with the presence of a predisposition to cholestasis that can be mild or even asymptomatic in the absence of clinical stress. Whether cholestasis, or a predisposition to cholestasis, is associated with other variables, such as specific MTM1 mutations, neonatal jaundice, and other hepatic abnormalities such as purpura, is currently unknown.

[0029] In relation to the currently proposed program, the clinical doses of AT132 used to date have been high (1E14 and 3E14 vg / kg), comparable to those used in other vector-based gene therapy programs, likely to compensate for suboptimal biodistribution and / or expression in the target tissue due to the use of a naturally occurring capsid that was not selected. According to the interim data from ASPIRO reported as of January 29, 2021, severe hepatobiliary events occurring during treatment were more frequent in the high-dose group (5 out of 17 participants in the high-dose group vs. 0 out of 6 in the low-dose group), but in the study participants reported in September 2021, it was shown that at least one subsequent event likely occurred in the low-dose group. According to recently published biopsy and autopsy data, study participants who experienced fatal liver events after treatment with AT132 were shown to have very high vector copy numbers (VCN) in the liver, both in absolute terms and compared to the heart and skeletal muscle. In contrast, neither MTM1 nor myotubulin was identified in liver tissue. Although not restricted to a mechanism of action, these findings suggest that hepatotoxicity may be driven by the capsid rather than the transgene, that dose is a factor, and that reducing liver exposure to the capsid may reduce the risk of vector-based gene therapy for boys with XLMTM. For example, other types of adverse events associated with vector-based gene therapy (such as complement activation and its sequelae) were not reported in the ASPIRO trial.

[0030] Adeno-associated virus vector AAV is a viral vector particularly suitable for the delivery of genetic material to mammalian cells. AAV is not known to cause disease in mammals and elicits a very mild immune response. Furthermore, AAV can infect cells at multiple stages, whether in a quiescent state or at a stage of the cell replication cycle. Advantageously, AAV DNA is not regularly inserted into the host genome at random sites, and for this reason this vector has low oncogenic properties.

[0031] AAV has been engineered to deliver various treatments, particularly for genetic disorders caused by single nucleotide polymorphisms (SNPs). Genetic diseases that have been studied in combination with AAV vectors include cystic fibrosis, hemophilia, arthritis, macular degeneration, muscular dystrophy, Parkinson's disease, congestive heart failure, and Alzheimer's disease. AAV can be used as a vector to deliver engineered nucleic acids to a host and utilize the host's own ribosomes to transcribe that nucleic acid into a desired protein. See, for example, West et al., Virology 160:38-47 (1987), U.S. Patent No. 4,797,368, WO93 / 24641, Kotin, Human Gene Therapy 5:793-801 (1994), and Muzyczka, J. Clin. Invest. 94:1351 (1994). AAV has some degree of defect in its replication and / or pathogenicity and may therefore be safer than adenoviral vectors. In some embodiments, AAV can integrate into a specific site on chromosome 19 of human cells without observable side effects. In some embodiments, the capacity of the AAV vector, its system, and / or AAV particles can be up to about 4.7 kb. The AAV vector or its system can include one or more of the engineered capsid polynucleotides described herein.

[0032] AAV is a small replication-defective non-enveloped virus that infects humans and other primate species and has a linear single-stranded DNA genome. Naturally occurring AAV serotypes exhibit liver tropism. As a result, transfection of non-liver tissues by conventional AAV vectors is hindered by the virus's natural liver tropism. Furthermore, since the liver acts to break down substances delivered to the subject, transfection of non-liver tissues by unmodified AAV vectors requires dosing at higher levels to provide a sufficient amount of virus to reach non-liver tissues beyond the liver. More than 30 naturally occurring AAV serotypes are available. There are many natural variants in the AAV capsid. Examples of AAV serotypes include, but are not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. AAV can be engineered using conventional molecular biology techniques, allowing these particles to be optimized, for example, for cell-specific delivery, for minimizing immunogenicity, for modulating stability and particle longevity, for efficient degradation, and for accurate delivery to the nucleus. AAV vectors can specifically target one or more types of cells by selecting an appropriate combination of AAV serotype, promoter, and delivery method.

[0033] Previous approaches to identify AAV sequences correlating with affinity relied on the comparison of existing highly related serotypes with different features, random domain swapping between unrelated serotypes, or consideration of higher-order structure to identify motifs that define liver affinity. For example, mapping of determinants of AAV affinity has been carried out by comparing highly related serotypes. One such example is the single amino acid change (E531K) between AAV1 and AAV6 that improves mouse liver transduction in AAV1. See Wu et al. (2006) J. Virol., 80(22):11393-7, which is incorporated herein by reference. Another example is the reciprocal domain swap between AAV2 and AAV8, which changes affinity but cannot define any robust specific tissue targeting motifs. See Raupp et al. (201) J. Virol., 86(l7):9396-408, which is incorporated herein by reference. Furthermore, overall consideration of structure highlights only the overall differences between better and worse liver transducers, which are more observational than actually useful. Nam et al (2007) J. Virol., 81(22):12260-71.

[0034] AAVs showing modified tissue affinity that can 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. WO2015 / 054653, PCT International Patent Application Publication No. WO2016 / 179496, PCT International Patent Application Publication No. WO2017 / 100791, and PCT International Patent Application Publication No. WO2019 / 217911, the entire contents of each of which are incorporated herein by reference.

[0035] An AAV vector or its system may contain one or more regulatory molecules such as promoters, enhancers, repressors, etc. In some embodiments, the AAV vector or its system may contain one or more polynucleotides capable of encoding one or more regulatory proteins. In some embodiments, the one or more regulatory proteins may be selected from Rep78, Rep68, Rep52, Rep40, their variants, and combinations thereof. In some embodiments, a muscle-specific promoter can drive the expression of the engineered AAV capsid polynucleotide.

[0036] An AAV vector or its system may contain one or more polynucleotides capable of encoding one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein. The engineered capsid protein may be able to assemble the protein shell (engineered capsid) of the AAV viral particle. The engineered capsid may have cell, tissue, and / or organ-specific affinity.

[0037] An 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 combination thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof. The AAV can be selected with respect to the cells to be targeted. For example, to target the brain and / or nerve cells, 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 heart tissue, AAV-4 can be selected; and to target the liver for delivery, AAV-8 can be selected. Thus, in some embodiments, an AAV vector or system thereof that can produce AAV particles capable of targeting the brain and / or nerve cells can be configured to generate AAV particles having serotype 1, 2, 5, or hybrid capsid AAV-1, AAV-2, AAV-5, or any combination thereof. In some embodiments, an AAV vector or system thereof that can produce AAV particles capable of targeting heart tissue can be configured to generate AAV particles having AAV-4 serotype. In some embodiments, an AAV vector or system thereof that can produce AAV particles capable of targeting the liver can be configured to generate AAV having AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21:75-80.

[0038] Even different serotypes can provide a certain level of cell, tissue, and / or organ specificity, but each serotype remains polyphilic and thus, it will be understood that using that serotype to target tissues with low efficiency of transduction by that serotype can result in tissue toxicity. Thus, in addition to achieving a certain level of tissue targeting ability through selection of a particular serotype of AAV, it will be understood that the affinity of the AAV serotype can also be modified by the engineered AAV capsids described herein. As described elsewhere herein, variants of wild-type AAV of any serotype can be generated via the methods described herein to determine having a particular cell-specific affinity, which affinity may be the same as or different from that of the reference wild-type AAV serotype. In some embodiments, it is possible to enhance the cell, tissue, and / or specificity of the wild-type serotype (e.g., making it more selective or specific for a particular cell type for which the serotype is already biased). 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 engineered AAV capsids and / or capsid protein variants of wild-type AAV-9 as described herein, the affinity for nerve cells may be reduced or eliminated, and / or muscle specificity may be enhanced, such that the nerve specificity is relatively reduced and thus appears to have enhanced specificity for muscle compared to wild-type AAV-9. As described above, by including engineered capsids and / or capsid protein variants of wild-type AAV serotypes, it is possible to have an affinity different from that of the reference wild-type AAV serotype. For example, engineered AAV capsids and / or capsid protein variants of AAV-9 can have specificity for tissues other than human muscle or brain.

[0039] In some embodiments, the AAV vector is a hybrid AAV vector or a system thereof. A hybrid AAV is an AAV that contains a genome in which elements from one serotype are packaged into a capsid derived from at least one different serotype. For example, if what is produced is rAAV2 / 5 and its production method is based on the transient transfection method without helper as discussed above, the first plasmid and the third plasmid (adeno helper plasmid) are considered to be the same as those discussed for rAAV2 production. However, the second plasmid, pRepCap, is considered to 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 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. The cell or tissue tropism exhibited by this AAV2 / 5 hybrid virus is expected to be the same as that of AAV5. It will be understood that wild-type hybrid AAV particles suffer from the same specificity problems as the non-hybrid wild-type serotypes discussed previously.

[0040] The advantages achieved by wild-type-based hybrid AAV systems can be combined with the improved customizable cell specificity achievable by engineered AAV capsids, which can be combined by generating hybrid AAVs that may include engineered AAV capsids described elsewhere herein. It will be understood that hybrid AAVs can contain engineered AAV capsids that contain a genome having elements from a serotype different from the reference wild-type serotype of which the engineered AAV capsid is a variant. For example, hybrid AAVs can be produced that contain an engineered AAV capsid that is a variant of the AAV-9 serotype used to package a genome containing components (e.g., rep elements) from the AAV-2 serotype. As in the case of the previously considered wild-type-based hybrid AAVs, the affinity of the resulting AAV particles is thought to be that of the engineered AAV capsid.

[0041] 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, the "gutless" AAV vector or system thereof can have cis-acting viral DNA elements linked to a heterologous sequence of interest (e.g., an engineered AAV capsid polynucleotide) and involved in genome amplification and packaging.

[0042] The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombinant and / or cloning methods or techniques can be used with the vectors described herein. Suitable recombinant 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.

[0043] The construction of recombinant AAV vectors is described in several publications, including U.S. Patent 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 these techniques and / or methods can be used and / or adapted to construct the AAV or other vectors described herein. AAV vectors are discussed elsewhere in this specification.

[0044] In some embodiments, the vector can have one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., 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.

[0045] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for the expression of one or more elements of the engineered AAV capsid systems described herein are as used in the aforementioned documents, e.g., International Patent Application Publication Nos. WO2021 / 050974 and WO2021 / 077000 and PCT International Application No. PCT / US2021 / 042812, the contents of which are incorporated herein by reference.

[0046] Additional AAV vectors are described in International Patent Application Publication No. WO2019 / 2071632, the contents of which are incorporated herein by reference.

[0047] Additional AAV vectors are described in International Patent Application Publications WO2020 / 086881 and WO2020 / 235543, the contents of each of which are incorporated herein by reference.

[0048] Additional AAV vectors are described in International Patent Application Publications WO2005 / 033321, WO2006 / 110689, WO2007 / 127264, WO2008 / 027084, WO2009 / 073103, WO2009 / 073104, WO2009 / 105084, WO2009 / 134681, WO2009 / 136977, WO2010 / 051367, WO2010 / 138675, WO2001 / 038187, WO2012 / 112832, WO2015 / 054653, WO2016 / 179496, WO2017 / 100791, WO2017 / 019994, WO2018 / 209154, WO2019 / 067982, WO2019 / 195701, WO2019 / 217911, WO2020 / 041498, WO2020 / 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. Patent No. 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,No. 087, US Patent Application Publication No. 2011-023353, US Patent Application Publication No. 2019-0015527, US Patent Application Publication No. 2020-155704, US Patent Application Publication No. 2017-0191079, US Patent Application Publication No. 2019-0218574, US Patent Application Publication No. 2020-0208176, US Patent Application Publication No. 2020-0325491, US Patent Application Publication No. 2019-0055523, US Patent Application Publication No. 2020-0385689, US Patent Application Publication No. 2009-0317417, US Patent Application Publication No. 2016-0051603, US Patent Application Publication No. 2016-00244783, US Patent Application Publication No. 2017-0183636, US Patent Application Publication No. 2020-0263201, US Patent Application Publication No. 2020-0101099, US Patent Application Publication No. 2020-0318082, US Patent Application Publication No. 2018-0369414, US Patent Application Publication No. 2019-0330278, US Patent Application Publication No. 2020-0231986, the contents of each of which are incorporated herein by reference.,

[0049] Capsid protein The capsid protein is the shell or coating of the virus that enables delivery to the host. Without this protein, the nucleic acid would be destroyed by the host without entering the host cell and initiating transcription and translation. The capsid protein may be in the natural conformation of a naturally occurring AAV or may be modified.,

[0050] In certain 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.,

[0051] In some embodiments, the polynucleotide encoding the engineered AAV capsid can be included in a polynucleotide configured to be an AAV genomic donor in an AAV vector system that can be used to generate the engineered AAV particles described elsewhere herein. In some embodiments, the polynucleotide encoding the engineered AAV capsid can be operably linked to a polyadenylation tail. In some embodiments, the polyadenylation tail can be an SV40 polyadenylation tail. In some embodiments, the polynucleotide encoding the AAV capsid 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 can be specific for muscle (e.g., cardiac muscle, skeletal muscle, and / or smooth muscle), neurons and supporting cells (e.g., astrocytes, glial cells, Schwann cells, etc.), adipose, spleen, liver, kidney, immune cells, cerebrospinal fluid cells, synovial fluid 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 promoters and constitutive promoters are discussed elsewhere herein, 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 the SPc5-12 synthetic promoter.

[0052] Engineered viral capsids, such as adeno-associated virus (AAV) capsids, that can be engineered to confer cell-specific affinities, such as muscle-specific affinities, to engineered viral particles are described herein. The engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids. The engineered capsids can be included in engineered viral particles (e.g., engineered lentiviral, retroviral, adenoviral, or AAV viral particles) and can confer cell-specific affinity, reduced immunogenicity, or both to the engineered viral particles. The engineered viral capsids described herein can include one or more of the engineered viral capsid proteins described herein. The engineered viral capsids described herein can include one or more of the engineered viral capsid proteins described herein that contain or are composed of a muscle-specific targeting moiety that contains the multimeric motifs described elsewhere herein.

[0053] Engineered viral capsids 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, an engineered lentiviral capsid polynucleotide, an engineered retroviral capsid polynucleotide, or an engineered adenoviral capsid polynucleotide. In some 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) can include a 3' polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

[0054] The engineered viral capsid can be a variant of the wild-type viral capsid. For example, in some embodiments, the engineered AAV capsid can be a variant of the wild-type AAV capsid. In some embodiments, the wild-type AAV capsid can be composed of VP1, VP2, VP3 capsid proteins or combinations thereof. In other words, the engineered AAV capsid can include one or more variants 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 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-type AAV capsid can be AAV-9. The engineered AAV capsid can have an affinity different from that of the reference wild-type AAV capsid.

[0055] The engineered viral capsid can contain 1 to 60 engineered capsid proteins. In some embodiments, the 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, the engineered viral capsid can contain 0 to 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.

[0056] In some embodiments, the engineered AAV capsid may contain 1 to 60 engineered capsid proteins. In some embodiments, the 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.

[0057] 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, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, the engineered AAV capsid can have a hexamer or heptamer 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 within a variable amino acid region of the viral capsid protein.

[0058] In some embodiments, the n-mer motif can be inserted between two amino acids within a variable amino acid region of the AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (beta B to beta I) and an alpha helix (alpha A) that are conserved in autonomous parvovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structurally variable regions (VRs) are present in surface loops that connect beta strands, which cluster to produce local changes on the capsid surface. AAV has 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 n-mer motifs can 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 can 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 combinations thereof. In some embodiments, the n-mer can be inserted between two amino acids in VR-III of the capsid protein.In some embodiments, the engineered capsid can have an n-mer inserted between any two consecutive amino acids between amino acids 262 and 269 of the AAV9 viral protein, 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, and between any two consecutive amino acids between amino acids 704 and 714. In some embodiments, the engineered capsid can have an n-mer inserted between amino acids 588 and 589 of the AAV9 viral protein. In some embodiments, the engineered capsid can have a heptameric motif inserted between amino acids 588 and 589 of the AAV9 viral protein. In other embodiments, the inserted motif is a decameric motif having a replacement of amino acids 586 - 588 and an insertion before 589. SEQ ID NO: 1 is a reference AAV9 capsid sequence for at least referring to the insertion sites described above. It will be understood that the n-mer can be inserted at similar positions in AAV viral proteins of other serotypes. As discussed above, in some embodiments, the n-mer can 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.

[0059] In some embodiments, the first 1, 2, 3, or 4 amino acids of the n-mer motif can replace 1, 2, 3, or 4 amino acids of the polypeptide preceding the insertion site into which it is inserted. 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 2-3, the first 3 amino acids shown can replace 1-3 amino acids in the polypeptide into which they can be inserted. As another non-limiting example, AAV can be used to insert one or more n-mer motifs, for example, into the AAV9 capsid polypeptide between amino acids 588 and 589, and this 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 understood that this principle can be applied to any other insertion situation and is not necessarily limited to insertion between residues 588 and 589 of the AAV9 capsid or equivalent positions in another AAV capsid. It will further be understood that in some embodiments, the amino acids in the polypeptide into which the n-mer motif is inserted are not replaced by the n-mer motif.

[0060] In some embodiments, an AAV capsid or other viral capsid or composition can be muscle-specific. In some embodiments, the muscle specificity of an 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 imparts a 3D structure to a domain or region of the engineered AAV capsid or other viral capsid or other composition such that the interaction of the virus particle or other composition containing the engineered AAV capsid or other viral capsid or other composition described herein with cell surface receptors and / or other molecules on the surface of muscle cells is increased or improved (e.g., increased affinity). In some embodiments, the cell surface receptor is the 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 a dimer thereof. In some embodiments, the cell surface receptor or molecule is a Vb6 integrin heterodimer.

[0061] In some embodiments, the muscle-specific engineered virus particles or other compositions described herein that contain a muscle-specific capsid, n-mer motif, or muscle-specific targeting moiety described herein have an increase in uptake, delivery rate, transduction rate, efficiency, amount, or combinations thereof in muscle cells as compared to other cell types and / or other virus particles (including but not limited to AAV) and other compositions that do not contain the muscle-specific n-mer motif of the invention.

[0062] The 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 non-human primates as described elsewhere herein and / or, for example, in U.S. Provisional Patent Applications Nos. 62 / 899,453, 62 / 916,207, 63 / 018,454, and 63 / 242,008. The first and second generation myoAAV capsids were further optimized in mice and non-human primates as described above to generate enhanced myoAAV capsids.

[0063] Tables 1 and 2 show the top hits of the enhanced muscle-specific n-mer motifs and their coding sequences in rank order in each table. The enhanced MyoAAV (eMyoAAV) capsid variants can transduce mouse muscle more effectively after systemic delivery compared to the first generation MyoAAV. The first and second generation myoAAV capsid variants are dependent on the αVβ6 integrin heterodimer for transduction into human primary myotubes.

[0064] Tables 3 and 4 show the top-ranked capsid variants produced in multiple rounds of directed evolution of capsid variants for skeletal muscle specificity. As shown in the above tables for the variant n-mer insert containing the P motif, the first three amino acids of the shown variant sequences are amino acids in which the amino acids corresponding to positions 596, 597, and 598 of the AAV9 capsid polypeptide have been replaced. Thus, for example, the P motif was inserted between amino acids 598 and 599 of the AAV9 vector.

[0065] AAV may further comprise vp1, vp2, and vp3 capsid proteins. The amino acid sequence of the vp1 capsid protein of the vector of the present invention may be selected from the sequences of Table 5, the amino acid sequence of the vp2 capsid protein may be selected from the sequences of Table 6, and / or the amino acid sequence of the vp3 capsid protein is selected from the sequences of Table 7.

[0066] Promoter The present invention may contain a muscle-specific promoter or another promoter. The promoter may be linked to a nucleic acid sequence such that transcription preferably occurs within muscle cells. The promoter region enables the host cell to transcribe the transgene only in the cell type and tissue or organ in which the desired protein is to be produced. Here, including a muscle-specific promoter is mainly because it is desirable that the protein is translated only in muscle cells. The specificity of the cell type into which the nucleic acid is delivered and thus in which the protein is translated is desirable because of the harmful effects that can occur from translating the nucleic acid in cells where the nucleic acid and thus the protein are not needed.

[0067] In some embodiments, the muscle-specific promoter results in an improvement in muscle cell ability, muscle cell specificity, a decrease in immunogenicity, or any combination thereof. As used herein, terms such as "muscle-specific", "muscle cell specificity", "muscle cell ability", "muscle cell-specific" refer to the improved specificity, selectivity, or ability of the muscle-specific targeting portion of the present invention for muscle cells and compositions incorporating said muscle-specific targeting portion, compared to non-muscle cells. In some embodiments, the cell specificity, or selectivity, or ability, or any combination thereof of the muscle-specific targeting portion or compositions incorporating the muscle-specific targeting portion described herein is at least 2-fold to at least 500-fold more specific, selective, and / or potent for muscle cells compared to non-muscle cells.

[0068] In some embodiments, the muscle cell-selective promoter utilized is MHCK7. MHCK7 is a 771-base pair long promoter that is small enough to be included in an AAV vector. MHCK7 directs expression in fast and slow skeletal muscle as well as cardiac muscle, and has low expression in the liver, lung, and spleen. The MHCK7 promoter is associated with high levels of expression in skeletal muscle including the diaphragm, and includes enhancers that drive expression particularly in the heart and skeletal muscle, but has little expression in off-target tissues. For example, the promoter can be the MHCK7 promoter having the nucleic acid sequence of SEQ ID NO: 2.

[0069] In some embodiments, the promoter described herein is inserted into an AAV protein (e.g., an AAV capsid protein) with reduced specificity (or no detectable, measurable, or clinically significant interaction) for one or more non-muscle cell types. 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, etc. In some embodiments, the non-muscle cell is a hepatocyte.

[0070] The term "operably linked" refers to the linkage of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other.

[0071] Further exemplary tissue-specific promoters include the U6 promoter sequence, MHCK7 promoter sequence, CK6 promoter sequence, tMCK promoter sequence, CK5 promoter sequence, MCK promoter sequence, HAS promoter sequence, MPZ promoter sequence, desmin promoter sequence, APOA2 promoter sequence, hAAT promoter sequence, INS promoter sequence, IRS2 promoter sequence, MYH6 promoter sequence, MYL2 promoter sequence, TNNI3 promoter sequence, SYN1 promoter sequence, GFAP promoter sequence, NES promoter sequence, MBP promoter sequence, or TH promoter sequence.

[0072] Muscle-specific promoters are described in International Patent Application Publication Nos. WO2020 / 006458 and WO2021 / 126880, the contents of each of which are incorporated herein by reference.

[0073] Additional muscle-specific promoters are described in U.S. Patent No. 9,133,482, U.S. Patent No. 10,105,453, U.S. Patent No. 10,301,367, U.S. Patent Application Publication No. 2020-0360534, PCT International Patent Application Publication Nos. WO2020 / 006458, WO2021 / 035120, WO2021 / 053124, and WO2021 / 077000, the contents of each of which are incorporated herein by reference.

[0074] Inducible and / or tissue-specific RNA polymerase II promoters have been previously described. RNA polymerase promoters are known in the art and are further described in U.S. Patent Application Publication No. 11,149,288, the content of which is incorporated herein by reference.

[0075] Exons that have undergone alternative splicing Aspects of the invention include exons that undergo alternative splicing and can be used in connection with viral vectors to effectively regulate the expression of the coding region of the MTM1 gene. In certain embodiments, the exon that undergoes alternative splicing regulates the coding region of interest in a conditional manner. The conditional manner means that the exon that undergoes alternative splicing regulates the expression of the coding region of interest in a manner that is controlled or influenced by one or more conditions including, but not limited to, environmental conditions, intracellular conditions, extracellular conditions, cell type (e.g., hepatocytes vs. muscle cells), gene expression patterns, or disease states. Thus, aspects of the invention include regulating the expression of the coding region of the MTM1 gene in a conditional manner by associating the expression of the coding region of interest with an exon cassette that undergoes alternative splicing. The exon that undergoes alternative splicing is described in PCT International Application No. PCT / US2022 / 017015, the entire contents of which are incorporated herein by reference.

[0076] In some embodiments, the exon cassette that undergoes alternative splicing includes 1, 2, 3, or 4 exons that undergo alternative splicing. In some other embodiments, the exon cassette that undergoes alternative splicing includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 exons that undergo alternative splicing. In some embodiments, when the exon cassette that undergoes alternative splicing includes two or more exons that undergo alternative splicing, the exons that undergo alternative splicing are adjacent. In some embodiments, when the exon cassette that undergoes alternative splicing includes two or more exons that undergo alternative splicing, the exons that undergo alternative splicing are not adjacent.

[0077] In some embodiments, the exon that has undergone alternative splicing is synthetic or recombinant. In some embodiments, the exon that has undergone alternative splicing is considered synthetic or recombinant because it has undergone one or more nucleic acid modifications compared to the exon that has undergone wild-type alternative splicing. The nucleic acid modification can be a substitution or deletion of one or more nucleotides that form the nucleic acid sequence of the exon that has undergone alternative splicing.

[0078] In some embodiments, the alternative exon contains an ATG start codon at its 3' end. As will be appreciated, in some embodiments, a wild-type or native alternative exon may contain an ATG start codon at its 3' end. In such embodiments, the alternative exon can contain nucleic acid modifications unrelated to the insertion of a heterologous start codon at the 3' end of the alternative exon. However, it will be further appreciated that in some embodiments, a wild-type or native alternative exon may not contain an ATG start codon at its 3' end. In such embodiments, the modification is made to the 3' end of the alternative exon to introduce a heterologous start codon, such that if the alternative exon is spliced in or retained in the spliced transcript, the downstream coding sequence is translated as a full-length protein. As will be appreciated, in some embodiments, depending on the sequence present at the 3' end of the wild-type or native alternative exon, one, two, or three nucleic acid substitutions may be required to introduce a heterologous ATG start codon at the 3' end of the alternative exon. In such embodiments, the 3' end of the exon that has undergone alternative splicing contains one nucleotide substitution compared to the exon that has undergone wild-type alternative splicing to form the ATG start codon. In such embodiments, the 3' end of the exon that has undergone alternative splicing contains two nucleotide substitutions compared to the exon that has undergone wild-type alternative splicing to form the ATG start codon. In such embodiments, the 3' end of the exon that has undergone alternative splicing contains three nucleotide substitutions compared to the exon that has undergone wild-type alternative splicing to form the ATG start codon.

[0079] In some embodiments, the modification comprises the insertion of a heterologous start codon or a portion of a heterologous start codon at the 3' end of an exon that has undergone alternative splicing (e.g., instead of 1 to 3 nucleic acids being substituted, 1 to 3 nucleic acids are added to the 3' end of an exon that has undergone alternative splicing to form an ATG start codon).

[0080] In some embodiments, the alternative exon contains one, two, or three nucleic acid substitutions at the 3′ end, resulting in a heterologous ATG start codon (e.g., when the exon that has undergone wild-type alternative splicing does not contain an ATG start codon at its 3′ end). In some such embodiments, the strength of the 5′ splice site of the alternative exon can be reduced compared to the strength of the 5′ splice site of the wild-type or native alternative exon. In such embodiments, one or more additional modifications can be made to the intron sequence located immediately downstream of the sequence containing the 3′ end of the alternative exon. In some embodiments, the first 10 nucleotides of the intron sequence located immediately downstream of the exon that has undergone alternative splicing contain one to five nucleotide substitutions compared to the native or wild-type intron sequence located immediately downstream of the native or wild-type alternative exon. In some embodiments, the first 10 nucleotides of the intron sequence located immediately downstream of the exon that has undergone alternative splicing contain one nucleotide substitution compared to the native or wild-type intron sequence located immediately downstream of the native or wild-type alternative exon. In some embodiments, the first 10 nucleotides of the intron sequence located immediately downstream of the exon that has undergone alternative splicing contain two nucleotide substitutions compared to the native or wild-type intron sequence located immediately downstream of the native or wild-type alternative exon. In some embodiments, the first 10 nucleotides of the intron sequence located immediately downstream of the exon that has undergone alternative splicing contain three nucleotide substitutions compared to the native or wild-type intron sequence located immediately downstream of the native or wild-type alternative exon. In some embodiments, the first 10 nucleotides of the intron sequence located immediately downstream of the exon that has undergone alternative splicing contain four nucleotide substitutions compared to the native or wild-type intron sequence located immediately downstream of the native or wild-type alternative exon. In some embodiments, the first 10 nucleotides of the intron sequence located immediately downstream of the exon that has undergone alternative splicing contain five nucleotide substitutions compared to the native or wild-type intron sequence located immediately downstream of the native or wild-type alternative exon.In some embodiments, one to five nucleotide substitutions restore or partially restore the strength of the 5' splice site of a selected exon compared to the strength of the 5' splice site of the native or wild-type selected exon.

[0081] Additionally or alternatively, in some embodiments, the modification includes disrupting or deleting any native start codons located 5' to the heterologous start codon. In some embodiments, if an exon cassette that undergoes alternative splicing includes two or more exons that undergo alternative splicing, any native start codons located 5' to the heterologous start codon of the most 5'-proximal exon that undergoes alternative splicing are disrupted or deleted. Additionally or alternatively, in some embodiments, the modification includes introducing a heterologous in-frame stop codon into an exon that undergoes alternative splicing, at least 50 nucleotides upstream of the next 5' splice junction. In some embodiments, an exon that undergoes alternative splicing is a nonsense-mediated decay (NMD) exon. In some embodiments, an NMD exon includes an in-frame stop codon at least 50 nucleotides upstream of the next 5' splice junction.

[0082] In some embodiments, an exon that undergoes alternative splicing is considered synthetic if it is located differently than native (e.g., ligated to a coding sequence that would not be ligated under wild-type or native conditions) compared to the wild-type alternatively spliced exon (e.g., is heterologous). In some embodiments, an exon that undergoes alternative splicing is considered synthetic if it (i) undergoes one or more nucleic acid modifications and (ii) is located differently than native compared to the wild-type alternatively spliced exon.

[0083] In some embodiments, the exon that has undergone alternative splicing is a regulatory exon. In some embodiments, a regulatory exon is an exon that is alternatively regulated (e.g., an exon known to be subject to an alternative splicing mechanism). It will be understood that alternative splicing is a process by which exons, or portions of exons or non-coding regions within a pre-mRNA transcript, are differentially joined or skipped to yield multiple protein isoforms encoded by a single gene.

[0084] Pharmaceutical composition Some embodiments of the present invention may include any acceptable form provided for AAV vectors. For example, the AAV vector can be provided to a subject in the form of a composition or formulation containing the AAV vector. The expression vectors of the present invention can be formulated and administered to treat various disease states by any means that results in contact between the active ingredient and the site of action of the agent in the body of the subject. The compositions, polynucleotides, polypeptides, particles, cells, vector systems, and combinations thereof described herein can be included in formulations such as pharmaceutical formulations. In some embodiments, formulations can be used to generate polypeptides and other particles comprising one or more muscle-specific targeting moieties described herein. In some embodiments, the formulation can be delivered to a subject in need thereof. In some embodiments, the components 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 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 to cells alone, or as an active ingredient such as in a pharmaceutical formulation. Thus, pharmaceutical formulations containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein are also described herein. In some embodiments, the pharmaceutical formulation can contain an effective amount of 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 or cell in need thereof.

[0085] In some embodiments, the amount of one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein contained in a pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg, based on the body weight of the subject in need thereof or the average body weight of a particular patient population to which the pharmaceutical formulation can be administered. The amount of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein in a pharmaceutical formulation can range from about 1 pg to about 10 g, 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 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 or more cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 or more cells per nL, μL, mL, or L.

[0086] In embodiments, when engineered AAV capsid particles are included in the formulation, the formulation can be from 1 transduction unit (TU) / mL to 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 1×10 11, 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , or 1×10 20 and may contain engineered AAV capsid particles up to 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , or 1×10 20 and may contain engineered AAV capsid particles up to 1×10

[0087] pharmaceutically acceptable carriers and auxiliary components and agents In embodiments, a 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 may further comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, sesame oil, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone, which do not react detrimentally with the active composition.

[0088] The pharmaceutical formulation can be sterilized and, if desired, can be mixed with adjuvants that do not react detrimentally with the active composition, such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts that affect osmotic pressure, buffering agents, coloring agents, flavoring agents and / or fragrances.

[0089] In some embodiments, the pharmaceutical formulations described herein can be dosage forms. The dosage forms can be adapted for administration by any suitable route. Suitable routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhalation, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavernous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intraventricular, and intradermal. Such dosage forms can be prepared by any method known in the art.

[0090] Dosage forms adapted for oral administration can be individual dosage units, such as capsules, pellets or tablets, powders or granules, solutions, suspensions in aqueous or non-aqueous liquids, edible foams or whips, or water-in-oil or oil-in-water liquid emulsions. In some embodiments, pharmaceutical formulations adapted for oral administration also include one or more agents that impart flavor, preserve, color, or aid in the dispersion of the pharmaceutical formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as a foam, spray, or liquid solution. In some embodiments, the oral dosage form can contain a therapeutically effective amount or an appropriate fraction thereof of a pharmaceutical formulation containing from about 1 ng to 1000 g of a composition containing one or more of the targeted effector fusion proteins and / or their complexes, or polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The oral dosage form can be administered to a subject in need thereof.

[0091] Where appropriate, the dosage forms described herein can be microencapsulated.

[0092] The dosage form can also be prepared to extend or sustain the release of any component. In some embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be components whose release is delayed. In other embodiments, the release of the auxiliary components included is delayed as needed. Suitable methods for delaying the release of a component include, but are not limited to, coating or encapsulating the component in a substance such as a polymer, wax, gel, etc. Sustained 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, devices, and processes for preparing sustained release dosage forms of tablets and capsules, as well as tablets and pellets, capsules, and granules. Sustained release can be for any time from about 1 hour to about 3 months or more.

[0093] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, acrylic polymers and copolymers, and methacrylic resins commercially available under the trade name EUDRAGIT (sold by Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0094] Coatings can be formed with various ratios of water-soluble polymers, water-insoluble polymers, and / or pH-dependent polymers, with or without water-insoluble / water-soluble non-polymeric excipients, to produce the desired release profile. Coatings can be applied to any dosage form (matrix or monolithic), including, but not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, and "as is ingredients" formulated in suspension form or sprinkle dosage form.

[0095] Dosage forms suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for the treatment of the eye or other external tissues, such as the mouth or skin, the pharmaceutical formulation is applied as a topical ointment or cream. When formulated as an ointment, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be formulated using a paraffinic or water-miscible ointment base. In some embodiments, the active ingredient can be formulated into a cream using an oil-in-water cream base or a water-in-oil base. Dosage forms suitable for topical administration in the mouth include lozenges, pastilles, and oral rinses.

[0096] Dosage forms adapted for nasal or inhaled administration include aerosols, solutions, suspension droplets, gels, or dry powders. In some embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein are contained in a dosage form adapted for inhalation in a reduced particle size form obtained or obtainable by micronization. In some embodiments, the particle size of the size-reduced (e.g., micronized) compound or its salt or solvate is defined by a D50 value of about 0.5 to about 10 microns, measured by suitable methods known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms in which the carrier or excipient is a liquid for administration as a nasal spray or nasal drops include aqueous or oily solutions / suspensions of the active ingredient (e.g., one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, and / or co-active agents), which can be produced by various types of metered-dose pressurized aerosols, nebulizers, or injectors.

[0097] In some embodiments, the dosage form can be an aerosol formulation suitable for administration by inhalation. In some of these embodiments, the aerosol formulation can contain a solution or fine suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or non-aqueous solvent. The aerosol formulation can be provided in a sterile form in a single or multiple dose quantity within a sealed container. In some of these embodiments, the sealed container is a single-dose or multi-dose nasal or aerosol dispenser (e.g., a metered-dose inhaler) equipped with a metering valve, which is intended to be discarded after the contents of the container have been used up.

[0098] When 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 hydrofluorocarbons. The dosage form of the aerosol formulation in other embodiments is contained in a pump sprayer. The pressurized aerosol formulation can also contain a solution or 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 cosolvents and / or modifiers incorporated, for example, to improve the stability and / or taste and / or particulate mass characteristics (amount and / or profile) of the formulation. Administration of the aerosol formulation can be once or several times a day, for example, 2, 3, 4, or 8 times a day, with one, two, or three doses delivered each time.

[0099] For some dosage forms suitable and / or adapted for inhalation administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, auxiliary active ingredients, and / or pharmaceutically acceptable salts thereof, such dosage forms can also contain a powder base, such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein are in a reduced particle size form. In further embodiments, performance improvers such as L-leucine or another amino acid, cellobiose octaacetate, and / or metal salts of stearic acid, such as magnesium stearate or calcium stearate.

[0100] In some embodiments, the aerosol dosage form can be configured such that each metered dose of the aerosol contains one or more of a predetermined amount of an active ingredient, such as one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.

[0101] Dosage forms adapted for vaginal administration can be provided as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas.

[0102] Dosage forms adapted for parenteral administration and / or for any type of injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intra-articular, intracavernous, gingival, subgingival, intrathecal, intravitreal, intracerebral, and intraventricular) can include aqueous and / or non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions that can include suspending and thickening agents. Dosage forms adapted for parenteral administration can be provided in single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The dosage can be lyophilized and reconstituted prior to administration by resuspending it in a sterile carrier. Immediate injection solutions and suspensions can, in some embodiments, be prepared from sterile powders, granules, and tablets.

[0103] Dosage forms adapted for ocular administration can, if desired, be made suitable for injection and can include aqueous and / or non-aqueous sterile solutions that can optionally contain antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the fluid contained within or surrounding the eye of the subject, and aqueous and non-aqueous sterile suspensions that can include suspending and thickening agents.

[0104] For some embodiments, the dosage form contains a predetermined amount of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein per unit dose. Thus, in some embodiments, such a predetermined amount of the unit dose can be administered once or multiple times a day. Such pharmaceutical formulations can be prepared by any of the methods well known in the art.

Example

[0105] (Example 1) Efficacy of KT-430 in MTM1 knockout mice (Test KTS1020) A novel composition named KT-430 was tested as a therapeutic candidate for the treatment of XLMTM. KT-430 contains a novel recombinant muscle affinity capsid called MyoAAV3.8 for the delivery of nucleic acids encoding the MTM1 protein. The MyoAAV3.8 capsid delivers the transgene described in SEQ ID NO: 4.

[0106] Recently, MTM1 gene replacement using first-generation MyoAAV capsids has been shown to improve survival, weight gain, and motor function when expressing the human MTM1 coding sequence in Mtm1 KO mice, demonstrating that the human protein is effective in mice. See Tabebordbar, 2021, Cell 184:4919-4938, the entire contents of which are hereby incorporated by reference. This is consistent with the high homology of MTM1 across eukaryotes. For example, the human protein is 92% and 96% identical to the mouse MTM1 protein and the canine MTM1 protein, respectively.

[0107] Recovery of functional myotubularin through AAV8-mediated gene therapy to express full-length canine MTM1 under the control of the desmin promoter was effective in the MTM1 p.N155K canine model. This AAV8-based vector, when administered IV at high doses of 2E14 vg / kg or greater, improved survival and led to sustained improvements in contractile muscle strength, motor and respiratory function, and histological abnormalities. These preclinical studies using AAV8-based gene therapy and the desmin promoter predicted the clinical efficacy of AT132 (NCT03199469; ASPIRO), a related AAV8-based gene therapy (AAV8-desmin-hMTM1), thus validating the predictability of these disease models and their pharmacological relevance.

[0108] In the present invention, KT-430 was tested for its ability to achieve effective levels of transgene expression in skeletal muscle at a dose one order of magnitude less than the lowest dose of AT132 used in ASPIRO, thanks to its design, particularly its novel muscle-affinity capsid, MyoAAV3.8. This dose may substantially reduce exposure of non-target tissues to KT-430, enable clinical efficacy (improvement in muscle strength and reduction in the need for mechanical ventilation), improve safety, and in particular, reduce the risk of hepatotoxicity in this population with large unmet medical needs.

[0109] Study Objectives The objective of the study was to evaluate the efficacy and biodistribution of KT-430 (MyoAAV3.8-MHCK7-hMTM1) in male MTM1 KO mice over a dose range (3E11, 1E12, and 3E12 vg / kg) for 10 weeks after a single IV injection.

[0110] Methods Mtm1 KO mice (B6;129S-Mtm1 tm1(Gt(OST290S77)Lex) was obtained from Taconic Biosciences and bred (Model#TF0892). This mouse model does not express MTM1 and, consistent with the previously published Mtm1 KO strain, exhibits a decrease in lifespan, myopathy, and motor abnormalities. Mice (n = 6 hemizygous males / group) were administered a single IV (retro-orbital) injection (10 mL / kg) of vehicle (PBS + 35 mM NaCl + 0.001% Pluronic® F68) or escalating doses of KT-430 at 4 weeks of age. To enable comparison with healthy mice, a group of WT male littermates was treated with vehicle in parallel. Mice were dosed at 4 weeks of age because previous studies have established that AAV-based gene replacement is effective when administered immediately after weaning but before the mice become moribund due to disease progression.

[0111] Four weeks after administration (8 weeks of age), when most of the vehicle-treated KO mice were still alive and functional tests including grip strength (Grip Strength Meter, Columbus Instruments), open field activity (Panlab's Harvard Apparatus Open-Field Arena / LE800SC), and spontaneous running wheel activity (Med Associates' Low Profile Wireless Running Wheel) could be performed, an interim efficacy assessment was conducted. Soon thereafter, most of the vehicle-treated mice required humane euthanasia due to disease progression. At 10 weeks after administration, the end of the study, another functional assessment was performed to evaluate the efficacy of the surviving mice by comparison with WT littermates. The mice were euthanized and the tibialis, biceps, and quadriceps muscles were weighed in pairs to evaluate muscle growth. Microscopic evaluation of the recovery of pathological abnormalities in the selected muscle tissues was also performed, which included quantification of central nuclei and fiber diameter, as well as staining with nicotinamide adenine dinucleotide (NADH) to evaluate organelle localization abnormalities. The heart and liver were also evaluated by light microscopy for potential toxicity. Tissues were evaluated for biodistribution, including vector copy number (VCN), hMTM1 transgene mRNA, and protein expression.

Table 5-37

[0112] The in vivo distribution of the vector genome was evaluated by digital droplet PCR (ddPCR) using Taqman primers / probes against the 3'-end of the hMTM1 coding sequence. The number of vector genomes was normalized to the number of diploid genomes using the mouse telomerase (Tert) reference gene. To measure the transgene mRNA, a TaqMan assay using the same primers against the 3'-end of the hMTM1 coding sequence was developed. The copy number of mRNA was quantified against a standard curve and normalized to the level of mouse Gapdh mRNA as a reference gene. Furthermore, the level of hMTM1 transgene mRNA was compared to the endogenous level of mouse Mtm1 determined from vehicle-treated WT littermates of the first group. The protein level of Mtm1 was determined by Western blot using an anti-Mtm1 antibody (Abnova).

[0113] Results Figures 1A - C show the results of KT-430 administration on survival and growth in Mtm1 KO mice. Mtm1 KO mice were evaluated for survival, body weight, and terminal muscle weight. Figure 1A, survival; Figure 1B, body weight; and Figure 1C, muscle weight of the quadriceps femoris and rectus femoris. Asterisks indicate statistical differences from vehicle-treated KO mice ( * p < 0.05; ** p < 0.01).

[0114] Treatment with KT-430 led to a dose-dependent increase in survival duration. All vehicle-treated KO mice had to be euthanized due to disease progression between 8 and 10 weeks of age, which was consistent with the reduced survival duration reported elsewhere in Mtm1 KO mice. In contrast, all 6 KO mice in the high-dose group (3E12 vg / kg) survived until the scheduled necropsy at week 10. In the low-dose group (3E11 vg / kg) and the intermediate-dose group (1E12 vg / kg), 2 out of 6 and 5 out of 6 animals survived until week 10, respectively. In KT-430-treated KO mice, a dose-dependent increase in muscle weight (quadriceps, tibialis) and body weight was also observed. Muscle and body weight in high-dose-treated KO mice were not significantly different from those of WT animals at the necropsy at week 10.

[0115] Figure 2 shows the results of KT-430 administration on muscle function in Mtm1 KO mice. Mtm1 KO mice were treated at 4 weeks of age, and motor function was evaluated 4 weeks after administration when vehicle-treated KO mice were still alive. Subsequently, a second evaluation of the mice was performed before necropsy at 10 weeks after administration. A–B) Mean peak force (Newton) of grip strength based on 5 repeated determinations per mouse. C–D) Spontaneous running wheel activity (average distance per day in kilometers) occurring over 7 days (4 weeks) or 8 days (10 weeks). E–F) Open field activity (total distance in centimeters) measured over 30 minutes in an open field arena. Asterisks indicate statistical differences from vehicle-treated KO mice ( * p < 0.05; ** p < 0.01; *** p < 0.001).

[0116] Vehicle-treated Mtm1 KO mice showed a dramatic decrease in grip strength, open field activity (distance traveled), and spontaneous running wheel activity (average daily running distance over 7–8 days) compared to WT littermates when measured at 8 weeks of age. Treatment with KT-430 brought about a dose-dependent improvement in these measures of motor function, but due to large inter-animal variability in mice, the results achieved statistical significance only at the high dose.

[0117] Vehicles-treated Mtm1 KO mice exhibit the expected pathological features of XLMTM, including decreased muscle fiber size, internal / central nucleation, and abnormal organelle localization in quadriceps and biceps muscles. Mtm1 KO mice treated with KT-430 showed dose-dependent recovery of these histological features in quadriceps and biceps muscles at doses of 1E12 vg / kg and 3E12 vg / kg, but no obvious improvement at the low dose of 3E11 vg / kg. This is consistent with the slight improvement in motor function at this dose level. The liver and heart were histologically normal at all tested doses of KT-430, further supporting the safety of KT-430. Also, no relevant changes were observed in serum chemistry test values, including alanine transaminase, aspartate transaminase, alkaline phosphatase, bilirubin, and creatine kinase, in KT-430-treated mice.

[0118] Vector copy number (VCN) The in vivo distribution of vector genomic DNA was evaluated in a subset of tissues from KT-430-treated mice surviving at week 10.

[0119] Figure 3 shows the in vivo distribution of KT-430 in Mtm1 KO mice. Abbreviations used in Figure 3 include KO = knockout, Mtm1 = mouse myotubularin gene, Tert = telomerase, WT = wild type. Vector copy number per diploid genome (mean ± standard deviation) in mice administered escalating doses of KT-430. Vector copy number is normalized to the mouse Tert gene (n = 3 - 6 mice / group).

[0120] Treatment with KT-430 resulted in a dose-dependent increase in vector genome per diploid genome in all tissues evaluated. The level in the liver at the effective dose of 3E12 vg / kg was approximately 1 vg / dg. The levels in muscle and heart were less than 0.1 vg / dg.

[0121] Transgene expression Figure 4 shows the dose-dependent expression of hMTM1 mRNA after KT-430 treatment in Mtm1 KO mice. Transgene mRNA levels (mean ± standard deviation) of the hMTM1 transgene from mice treated with increasing doses of KT-430 (n = 3 - 5 mice / group), compared to endogenous mouse Mtm1 mRNA, were measured in vehicle-treated WT mice. All data are normalized to the level of mouse Gapdh.

[0122] KT-430 treatment resulted in a dose-dependent increase in hMTM1 transgene mRNA in muscle and heart at intermediate and high doses of 1E12 and 3E12 vg / kg. Compared to the endogenous mouse Mtm1 mRNA level, a dose of 1E12 vg / kg resulted in hMTM1 mRNA levels that were approximately the same as physiological levels in muscle and heart. The high dose of 3E12 vg / kg resulted in transgene mRNA levels that exceeded physiological levels (7 - 16-fold normal) in muscle and heart. The low dose of 3E11 vg / kg produced detectable but very low levels of transgene mRNA, which were below physiological levels. Interestingly, at the high dose of 3E12 vg / kg in the liver, physiological levels of transgene mRNA were produced, indicating that the MHCK7 promoter has weak activity in the liver.

[0123] Figure 5 shows the dose-dependent expression of MTM1 protein after KT-430 treatment in Mtm1 KO mice. hMTM1 protein levels (mean ± standard deviation) from mice treated with increasing doses of KT-430 (n = 3 - 5 mice / group), compared to endogenous mouse MTM1 in WT animals.

[0124] As predicted based on mRNA levels, KT-430 caused a dose-dependent increase in hMTM1 protein expression in muscle, heart, and liver. At 3E11 vg / kg, no significant MTM1 expression was observed in any tissue, but in muscle and heart, at 1E12 vg / kg, MTM1 expression below physiological levels (less than 100% of normal) was observed, and at 3E12 vg / kg, expression above physiological levels (above 100% of normal) was observed.

[0125] Conclusion In summary, these results demonstrate that KT-430 treatment results in dose-dependent improvements in survival, body weight gain, and motor function. KT-430 is fully effective at a dose of 3E12 vg / kg, consistent with achieving at least 100% of normal MTM1 expression in muscle. The intermediate dose of 1E12 vg / kg showed some evidence of efficacy based on trends in survival and improvements in body weight, muscle weight, and motor function. This is consistent with less expression below the physiological levels of hMTM1 mRNA and protein. Based on efficacy and safety data, and consistent MTM1 protein expression in muscle at 10 weeks post-administration, treatment with KT-430 does not appear to be associated with an anti-transgene immune response or hepatotoxicity. These results support the biological activity of KT-430 and demonstrate that MTM1 gene replacement can restore disease symptoms when expressed at physiological levels. This result also supports the safety of KT-430.

[0126] Incorporation by reference References and citations to other documents such as patents, patent applications, patent publications, journals, books, papers, web content, etc. are made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0127] Equivalents In addition to what is shown and described in this specification, various modifications of the invention and many further embodiments thereof will become apparent to those skilled in the art from the entire contents of this document, including references to scientific and patent literature cited herein. The subject matter of this specification includes important information, exemplification and guidance that can be adapted for the practice of the invention in its various embodiments and their equivalents. Additional array

Claims

1. Capsid proteins comprising at least one modification that results in preferential targeting of muscle tissue by adeno-associated virus (AAV) vectors, and Nucleic acid encoding the full-length MTM1 protein An AAV vector comprising, wherein the modification comprises a peptide having one of the amino acid sequences of SEQ ID NOs. 381-556, 6-180, and 757-1249.

2. The AAV vector according to claim 1, wherein the peptide has one amino acid sequence from sequence numbers 381 to 556.

3. The AAV vector according to claim 2, wherein the peptide has the amino acid sequence of SEQ ID NO:

381.

4. The AAV vector according to claim 2, wherein the peptide has the amino acid sequence of SEQ ID NO:

397.

5. The AAV vector according to claim 2, wherein the peptide has the amino acid sequence of SEQ ID NO:

398.

6. The AAV vector according to claim 2, wherein the peptide has the amino acid sequence of SEQ ID NO:

382.

7. The AAV vector according to claim 2, wherein the peptide has the amino acid sequence of SEQ ID NO:

386.

8. The AAV vector according to claim 1, wherein the peptide has one amino acid sequence from SEQ ID NOs: 6 to 180.

9. The AAV vector according to claim 1, wherein the modification includes an insertion between any two consecutive amino acids in the AAV9 capsid polypeptide 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, or an insertion at a similar position in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh. 74, or AAV rh. 10 capsid polypeptide.

10. The AAV vector according to claim 1, wherein the modification includes substitution of amino acids 586-588 and insertion between amino acids 588 and 589 in the AAV9 capsid polypeptide, or substitution and insertion at similar positions in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh. 74, or AAV rh. 10 capsid polypeptide.

11. The modification includes the substitution of amino acids 586-588 and the insertion between amino acids 588 and 589 in the AAV9 capsid polypeptide having the amino acid sequence of SEQ ID NO:

1. (i) The substitution comprises one of three N-terminal residues from sequence numbers 381-556, 6-180, and 757-1249, (ii) The AAV vector according to claim 1, wherein the insertion comprises one of seven C-terminal residues from sequence numbers 381-556, 6-180, and 757-1249.

12. (i) The substitution comprises the 3 N-terminal residue of Sequence ID No. 381, (ii) The insertion includes the 7 C-terminal residue of SEQ ID NO: 381, The AAV vector according to claim 11.

13. (i) The substitution comprises the 3 N-terminal residue of Sequence ID No. 397, (ii) The insertion includes the 7 C-terminal residue of Sequence ID No. 397, The AAV vector according to claim 11.

14. (i) The substitution comprises the 3 N-terminal residue of SEQ ID NO: 398, (ii) The insertion includes the 7 C-terminal residue of SEQ ID NO: 398, The AAV vector according to claim 11.

15. (i) The substitution comprises the 3 N-terminal residue of Sequence ID No. 382, (ii) The insertion includes the 7 C-terminal residue of SEQ ID NO: 382, The AAV vector according to claim 11.

16. (i) The substitution comprises the 3 N-terminal residue of Sequence ID No. 386, (ii) The insertion includes the 7 C-terminal residue of SEQ ID NO: 386, The AAV vector according to claim 11.

17. AAV contains vp1 capsid protein, vp2 capsid protein, and vp3 capsid protein. The amino acid sequence of the vp1 capsid protein is selected from one of sequence numbers 1250 to 1357. The amino acid sequence of the vp2 capsid protein is selected from any one of sequence numbers 1358 to 1465, and / or The AAV vector according to claim 1, wherein the amino acid sequence of the vp3 capsid protein is selected from any one of sequence numbers 1466 to 1494.

18. The amino acid sequence of the vp1 capsid protein has the sequence of SEQ ID NO: 1260, The amino acid sequence of the vp2 capsid protein has the sequence of sequence number 1436, The amino acid sequence of the vp3 capsid protein has the sequence of sequence number 1479. The AAV vector according to claim 17.

19. The AAV vector according to claim 1, further comprising at least one modification of the capsid protein that results in a decrease in the liver affinity of the AAV vector.

20. The AAV vector according to claim 1, wherein the nucleic acid encoding the full-length MTM1 protein is operably linked to a muscle-specific promoter.

21. The AAV vector according to claim 20, wherein the muscle-specific promoter is the MHCK7 promoter.

22. The AAV vector according to claim 20, wherein the nucleic acid encoding the full-length MTM1 protein comprises a selectively spliced ​​exon cassette downstream of the muscle-specific promoter, and the selectively spliced ​​exon cassette comprises an ATG start codon at the 3' end of the cassette.

23. The AAV vector according to claim 22, wherein the exon cassette that has undergone alternative splicing includes skeletal muscle-specific exons.

24. The AAV vector according to claim 23, wherein the exon cassette that has undergone alternative splicing promotes skeletal muscle-specific expression of the nucleic acid.

25. A pharmaceutical composition for use in the treatment of X-linked myotubular myopathy (XLMTM) in subjects suffering from X-linked myotubular myopathy, comprising the AAV vector according to any one of claims 1 to 24.

Images