JP2025537282A - Designer extracellular vesicles for targeted delivery to muscle cells - Google Patents

Abstract

Disclosed herein are designer extracellular vesicles (EVs) targeted to muscle cells. For example, in some embodiments, these EVs are modified with fusion proteins comprising NHERF1, NHERF2, the E8 fragment of laminin and an exosomal or lysosomal transmembrane protein, or combinations thereof. In some embodiments, these EVs can be used to deliver diagnostic and/or therapeutic cargoes to muscle cells in a subject in need of such cargo. In some embodiments, these EVs incorporate a DUX4 silencing oligonucleotide to treat facioscapulohumeral muscular dystrophy (FSHD) in a subject. For example, in some embodiments, the therapeutic cargo is a DUX4 silencing oligonucleotide.
[Selected Figure] Figure 1A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/383,188, filed November 10, 2022, the contents of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING This application contains a Sequence Listing having 11,940 bytes, submitted in ST.26 format entitled "321501-2660 Sequence Listing," created on August 24, 2023. The contents of the Sequence Listing are incorporated herein in their entirety.

Facioscapulohumeral muscular dystrophy (FSHD) is a rare, progressive, and disabling disease for which there is no approved treatment. The disease is characterized by progressive skeletal muscle weakness, beginning with weakness in the face, shoulders, arms, and trunk muscles and progressing to weakness throughout the lower body. Skeletal muscle weakness can lead to significant physical limitations, such as an inability to smile and difficulty using the arms for activities, and many patients eventually become wheelchair-dependent for daily mobility.

FSHD is caused by abnormal expression of DUX4 in skeletal muscle, resulting in inappropriate production of the DUX4 protein. Normally, DUX4 gene expression is restricted to early embryonic development, after which the DUX4 gene is silenced. In FSHD patients, genetic mutations result in the release of DUX4 gene silencing. This results in muscle death and replacement with fat, leading to a loss of skeletal muscle strength and progression of the disorder.

Disclosed herein are designer extracellular vesicles (EVs) that target skeletal muscle cells and precursor muscle cells, such as myoblasts and satellite cells, and are engineered to express a fusion protein comprising NHERF1, NHERF2, an E8 fragment of laminin, and an exosomal or lysosomal transmembrane protein. For example, in some embodiments, these designer EVs are derived from somatic cells that have been genetically engineered to express NHERF1, NHERF2, or an E8 fragment of laminin associated with an exosomal or lysosomal transmembrane protein. In some other embodiments, the designer EVs are derived from muscle cells, including precursor supporting cells (e.g., satellite cells and myoblasts), that have been engineered to express NHERF1, NHERF2, or an E8 fragment of laminin associated with an exosomal or lysosomal transmembrane protein, which is expected to enhance endogenous targeting for muscle tissue. In some embodiments, the method involves functionalizing EVs isolated from somatic cells, including muscle cells and their precursor supporting cells, with NHERF1, NHERF2, or an E8 fragment of laminin associated with exosomal or lysosomal proteins. NHERF1 and/or NHERF2 target the CD34 receptor in myogenic tissue, while the E8 fragment of laminin targets the α7β1 integrin present in the sarcolemma.

In some embodiments, these EVs can be used to deliver diagnostic and/or therapeutic cargo to muscle cells in a subject in need of that cargo.

Accordingly, further disclosed herein are methods for treating any of the diseases or conditions associated with muscle cells, such as Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy type 1C (LGMD1C), facioscapulohumeral muscular dystrophy (FSHMD), Becker muscular dystrophy (BMD), amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, myasthenia gravis, and myopathy. Accordingly, the EVs disclosed herein can be used to treat one or more of these conditions.

In some embodiments, these EVs incorporate DUX4 RNAi, p38α, p38β (Mapk14) (which also suppress DUX4 expression), myostatin, and NF-κB/p65, and can therefore be used to treat the disease in a subject. Accordingly, further disclosed herein are methods for treating FSHD, DMD, and LGMD1C in a subject, comprising modifying the subject's cells to produce therapeutic EVs that target muscle cells and deliver a therapeutic cargo.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the following detailed description. Other features, objects, and advantages of the invention will be apparent from the detailed description and drawings, and from the claims.

These figures show the isolation and characterization of modified EVs carrying shRNA p38b.

Figure 1 shows the isolation and characterization of modified EVs incorporating shRNA p38b.Representative immunofluorescence images of donor cells (in this case, primary mouse embryonic fibroblasts (PMEFs)) showing positive expression of the GFP reporter contained in the expression plasmid used to modify EVs 24 hours after electroporation. Figure 1 shows the isolation and characterization of modified EVs incorporating shRNA p38b. qRT-PCR results showing strong upregulation of GFP in donor cells 24 hours after electroporation. Figure 1 shows the isolation and characterization of modified EVs incorporating shRNA p38b. PCR products showing positive expression of lentiviral plasmid in scrambled (control) and shRNA p38b-transfected donor cells. Figure 1 shows the isolation and characterization of modified EVs incorporating shRNA p38b. Gene-level expression of GFP transcripts confirms that shRNA p38b is efficiently integrated into the modified EVs. Figure 1 shows the isolation and characterization of modified EVs incorporating shRNA p38b. Nanotracking particle analysis shows particle concentrations for modified EVs incorporating shRNA p38b, sham EVs, and naive EVs (released from non-transfected donor cells). Figure 1 shows the isolation and characterization of modified EVs incorporating shRNA p38b. Nanotracking particle analysis shows the particle size distribution for modified EVs incorporating shRNA p38b, sham EVs, and naive EVs (released from non-transfected donor cells). Isolation and characterization of modified EVs incorporating shRNA p38b are shown. PCR products showing positive expression of the lentiviral backbone in scrambled and shRNA p38b-incorporated modified EVs. (One-way ANOVA: *p<0.05, n=3).

Before describing the present disclosure in more detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is used only for the purpose of describing particular embodiments and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

Where a range of numerical values is provided, unless the context clearly dictates otherwise, it is understood that each intervening value (to the tenth of the unit of the lower limit) between the upper and lower limits of that range, as well as any other value or intervening value stated in the stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, except where there is a specifically excluded limit in the stated range. When the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described below.

All publications and patents cited herein are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for the sole purpose of indicating its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may require independent confirmation.

As will be apparent to one of ordinary skill in the art upon reading this disclosure, the individual embodiments described and illustrated herein have individual components and features that may be readily separated or combined with any of the features of several other embodiments without departing from the scope or spirit of the present disclosure. Any methods described can be performed in the order described or in any other order that is logically possible.

Embodiments of the present disclosure employ techniques of chemistry, biology, and the like that are within the skill of the art, unless otherwise indicated.

The following examples are presented to fully disclose and describe to those of ordinary skill in the art how to practice the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but please note that some errors and variations may have occurred. Unless otherwise indicated, parts are parts by weight, temperatures are in °C, and pressures are at or near atmospheric. Standard temperature and pressure are defined as 20°C and 1 atmosphere.

Before describing embodiments of the present disclosure in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reactants, or manufacturing steps, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is also to be understood that the present disclosure may allow steps to be executed in differing order where this is logically possible.

Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

DEFINITIONS The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., a mammal. Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

The term "therapeutically effective" refers to an amount of a composition used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration does not necessarily mean elimination, but may suffice to reduce or alter.

The term "pharmaceutically acceptable" refers to compounds, substances, compositions, and/or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, with a reasonable benefit/risk ratio.

The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. The term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure a disease, condition, or disorder; preventative treatment, i.e., treatment directed at minimizing or partially or completely suppressing the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed at ameliorating the associated disease, condition, or disorder.

The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to the native or control level.

The term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. Polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphitidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodation, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to proteins such as arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, symbols, or words that represent amino acid residues. Amino acid abbreviations used herein are the conventional single-letter codes for amino acids and are represented as follows: A: alanine, B: asparagine or aspartic acid, C: cysteine, D: aspartic acid, E: glutamic acid, F: phenylalanine, G: glycine, H: histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N: asparagine, P: proline, Q: glutamine, R: arginine, S: serine, T: threonine, V: valine, W: tryptophan, Y: tyrosine, Z: glutamine or glutamic acid.

As used herein, the term "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide capable of hybridizing to a complementary nucleic acid via Watson-Crick base pairing, whether DNA or RNA, or a DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense. Nucleic acids may also contain nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). Specifically, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

As used herein, a "nucleotide" is a molecule containing a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate and sugar moieties to form an internucleoside linkage. The term "oligonucleotide" is sometimes used to refer to a molecule containing two or more nucleotides linked together. The base moiety of a nucleotide can be adenine-9-yl (A), cytosin-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. Non-limiting examples of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

A nucleotide analog is a nucleotide that contains some type of modification to the base, sugar, and/or phosphate moiety. Modifications to nucleotides are well known in the art and may include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications to the sugar or phosphate moiety.

Nucleotide substitutes are molecules that have similar functional properties to nucleotides but do not contain a phosphate moiety, such as peptide nucleic acids (PNAs). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through moieties other than the phosphate moiety. Nucleotide substitutes can conform to a double-helix structure when interacting with an appropriate target nucleic acid.

The term "vector" or "construct" refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence is linked. The term "expression vector" includes any vector (e.g., a plasmid, cosmid, or phage chromosome) that contains a genetic construct in a form suitable for expression by the cell (e.g., linked to transcriptional control elements). "Plasmid" and "vector" are used interchangeably, as a plasmid is a commonly used form of vector. Furthermore, the invention is intended to include other vectors that serve equivalent functions.

The term "operably linked" refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operably linking DNA to a transcriptional regulatory element refers to a physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

As used herein, the percent sequence identity of a given nucleotide or amino acid sequence C to, with, or relative to a given nucleic acid sequence D (which may also be expressed as a given sequence C having or comprising a certain percent sequence identity to, with, or relative to a given sequence D) is defined hereinafter as:
100 x fraction W/Z
It is calculated as follows:
where W is the number of nucleotides or amino acids scored by a sequence alignment program as identical matches in that program's alignment of C and D, and Z is the total number of nucleotides or amino acids in D. It will be understood that if the length of sequence C is not equal to the length of sequence D, then the % sequence identity of C to D will not equal the % sequence identity of D to C. Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.

"Specifically hybridize" means that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base pairs with) a substantially complementary nucleic acid (e.g., a c-met nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

As used herein, the term "stringent hybridization conditions" means that hybridization typically occurs when there is at least 95%, preferably at least 97%, sequence identity between the probe and target sequence. An example of stringent hybridization conditions is overnight incubation in a solution containing 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrier DNA (such as salmon sperm DNA), followed by washing the hybridization support in 0.1x SSC at approximately 65°C. Other hybridization and washing conditions are well known and are exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), particularly Chapter 11.

"Control elements" or "regulatory sequences" are untranslated regions, such as vector enhancers, promoters, and 5' and 3' untranslated regions, that interact with host cell proteins to effect transcription and translation. Such elements can vary in their strength and specificity.

A "promoter" is generally a sequence(s) of DNA that functions when in a relatively fixed location with respect to the transcription start site. A "promoter" contains core elements required for basic interaction of RNA polymerase and transcription factors, and can contain upstream elements and response elements.

"Enhancer" generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Furthermore, enhancers can be found within introns as well as within the coding sequence itself. They are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Like promoters, enhancers also often contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.

An "endogenous" enhancer/promoter is one that is naturally associated with a given gene in the genome. An "exogenous" or "heterologous" enhancer/promoter is one that is juxtaposed to a gene by genetic engineering (i.e., molecular biological techniques) so that transcription of that gene is directed by the associated enhancer/promoter.

Myocyte-targeted extracellular vehicles (EVs)
Disclosed herein are muscle cell-targeting EVs that can incorporate therapeutic and/or diagnostic cargo. In some embodiments, the method includes harvesting muscle cells from a subject and isolating EVs that enhance homing to muscle tissue. In other embodiments, the method includes modifying the subject's cells to produce therapeutic EVs. In some embodiments, the method includes harvesting ex vivo-produced EVs and incorporating therapeutic cargo into them. Finally, in some embodiments, the method includes functionalizing the EVs after synthesis.

Modification of Patient Cells to Produce Therapeutic EVs Methods of reprogramming cells of a subject into EV-producing cells are disclosed, the method comprising intracellular delivery to the cells of a polynucleotide comprising a nucleic acid sequence encoding a muscle cell ligand and, optionally, a therapeutic cargo. In some embodiments, the cells can be any cell in the subject capable of producing EVs, including (but not limited to) skin cells (e.g., fibroblasts, keratinocytes, skin stem cells), adipocytes, dendritic cells, peripheral blood mononuclear cells (PBMCs), pancreatic cells (e.g., ductal epithelial cells), liver cells (e.g., hepatocytes), and immune cells (e.g., T cells, macrophages, myeloid-derived suppressor cells).

In some embodiments, the method includes transfecting cells of a subject with an expression vector encoding NHERF1 (SLC9A3 regulator 1), NHERF2 (SLC9A3 regulator 2), a laminin E8 fragment, or any combination thereof. In some embodiments, the method includes post-synthesis functionalization of EVs with a fusion protein and/or ligand, such as an E8 fragment of laminin.

In some embodiments, the SLC9A3 regulator 1 (SLC9A3R1) cDNA has the following nucleic acid sequence:
AGACGCCGCGCGGGGCGGGGATTGGTCTGTGGTCCTCTCTCGGCTCCTCGCGGCTCGCGGCG GCCGACGGTTCCTGGGACACCTGCTTGCTTGGCCCGTCCGGCGGCTCAGGGCTTCTCTGCTGC GCTCCCGGTTCGCTGGACGGGAAGAAGGGCTGGGCCGTCCCGTCCCGTCCCCATCGGAACCC CAAGTCGCGCCGCTGACCCGTCGCAGGGCGAGATGAGCGCGGACGCAGCGGCCGGGGCGCCCC TGCCCCGGCTCTGCTGCCTGGAGAAGGGTCCGAACGGCTACGGCTTCCACCTGCACGGGGAG AAGGGCAAGTTGGGCCAGTACATCCGGCTGGTGGAGCCCGGCTCGCCGGCCGAGAAGGCGGGG CTGCTGGCGGGGACCGGCTGGTGGAGGTGAACGGCGAAACGTGGAGAAGGAGACCCACCA GCAGGTGGTGAGCCGCATCCGCGCCGCACTCAACGCCGTGCGCCTGCTGGTGGTCGACCCCGA GACGGACGAGCAGCTGCAGAAGCTCGGCGTCCAGGTCCGAGAGGAGCTGCTGCGCGCCCAGG AAGCGCCGGGGCAGGCCGAGCCGCCGGCCGCCGCCGAGGTGCAGGGGGCTGGCAACGAAAATG AGCCTCGCGAGGCCGACAAGAGCCACCCGGAGCAGCGCGAGCTTCGGCCTCGGCTCTGTACC ATGAAGAAGGGCCCCAGTGGCTATGGCTTCAACCTGCAGCCGACAAGTCCAAGCCAGGCCAG TTCATCCGGTCAGTGGACCCAGACTCCCCGGCTGAGGCTTCAGGGCTCCGGGCCCAGGATCG CATTGTGGAGGTGAACGGGGTCTGCATGGAGGGGAAGCAGCATGGGGACGTGGTGTCCGCCAT CAGGGCTGGCGGGGACGAGACCAAGCTGCTGGTGGTGGACAGGGAAAACTGACGAGTTCTTCA AGAAATGCAGAGTGATCCCATCTCAGGAGCACCTGAATGGTCCCCTGCCTGTGCCCTTCACCA ATGGGGAGATACAGAAGGAGAACAGTCGTGAAGCCCTGGCAGAGGCAGCCTTGGAGAGCCCC AGGCCAGCCCTGGTGAGATCCGCCTCCAGTGACACCAGCGAGGAGCTGAATTCCCAAGACAGC CCCCCAAACAGGACTCCACAGCGCCCTCGTCTACCTCCTCCTCCGACCCCCATCCTAGACTT CAACATCTCCCTGGCCATGGCCAAAGAGAGGGCCCACCAGAAACGCAGCAGCAAACGGGCCCC GCAGATGGACTGGAGCAAGAAAAACGAACTCTTCAGCAACCTCTGAGCGCCCTGCTGCCACC CAGTGACTGGCAGGGCCGAGCCAGCATTCCACCCCACCTTTTTCCTTCTCCCCAATTACTCCC CTGAATCAATGTACAAATCAGCACCCACATCCCCTTTCTTGACAAATGATTTTTCTAGAGAA CTATGTTCTTCCCTGACTTTTAGGGAAGGTGAATGTGTTCCCGTCCTCCCGCAGTCAGAAAGGA GACTCTGCCTCCCTCCTCCTCACTGAGTGCCTCATCCCTACCGGGTGTCCCTTTGCCACCCTG CCTGGGACATCGCTGGAACCTGCACCATGCCAGGATCATGGGACCAGGCGAGAGGGCACCCTC CCTTCCTCCCCCATGTGATAAAATGGGTCCAGGGCTGATCAAGAACTCTGACTGCAGAACTG CCGCTCTCAGTGGACAGGGCATCTGTTACCCTGAGACCTGTGGCAGACACGTCTTGTTTTCAT TTGATTTTTTGTTAAGAGTGCAGTATTGCAGAGTCTAGAGGAATTTTGTTTCCTTGATTAAC ATGATTTTCCTGGTTGTTACATCCAGGGCATGGCAGTGGCCTCAGCCTTAAAACTTTGTTCCT ACTCCCCACCCTCAGCGAACTGGGCAGCACGGGGAGGGTTTGGCTACCCCTGCCCATCCCTGA GCCAGGTACCACCATTGTAAGGAAACACTTTCAGAAAATTCAGCTGGTTCCTCCAAA (SEQ ID NO: 1)

In some embodiments, the SLC9A3 regulator 1 (SLC9A3R1) mRNA encodes the following amino acid sequence:
MSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKGKLGQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEKETHQQVVSRIRAALNAVRLLLVV DPETDEQLQKLGVQVREELLRAQEAPGQAEPPAAAAEVQGAGNENEPREADKSHPEQRELRPRLCTMKKGPSGYGFNLHSDKSKPGQFIRSV DPDSPAEASGLRAQDRIVEVNGVCMEGKQHGDVVSAIRAGGDETKLLVVDRETDEFFKKCRVIPSQEHLNGPLPVPFTNGEIQKENSREALAEAALESPRPALVRSASSDTSEELNSQDSPPKQDSTAPSSTSSSDPILDFNISLAMAKERAHQKRSSKRAPQMDWSKKNELFSNL (SEQ ID NO: 2)

In some embodiments, the SLC9A3 regulator 2 (SLC9A3R2) cDNA has the following nucleic acid sequence:
GAACAGGAGCCGCCGCTGAAGCCACCGCCGGGTGCCCAGCGCCGCCGCCGCCCCCGAGCTCCCCCGC GCCCCTGCCCGCGGGCGGCCGGTGGGCAGCGGGCGCCATGGCCGCGCCGGAGCCGCTGCGGCCGCGCC TGTGCCGCTTGGTGCGCGGAGAGCAGGGCTACGGCTTCCACCTGCACGGCGAGAAGGGCCGCCGCG GCAGTTCATCCGGCGCGTGGAACCCGGTTCCCCCGCCGAGGCCGCCGCGCTGCGCGCTGGGGACCGCC TGGTCGAGGTCAACGGCGTCAACGTGGAGGGCGAGACGCACCACCAGGTGGTGCAAGGATCAAGGC TGTGGAGGGGCAGACTCGGCTGCTGGTGGTGGACCAGGAGACAGATGAGGAGCTCCGCCGGCGGCAGC TGACCTGTACCGAGGAGATGGCCCAGCGAGGGCTCCCACCCGCCCACGACCCCTGGGAGCCGAAGCCA GACTGGGCACACACCGGCAGCCACAGCTCCGAAGCTGGCAAGAAGGATGTCAGTGGGCCCCTGAGGGGA GCTGCGCCCTCGGCTCTGCCACCTGCGAAAGGGACCTCAGGGCTATGGGTTCAACCTGCATAGTGAC AAGTCCCGGCCCGGCCAGTACATCCGCTCTGTGGACCCGGGCTCACCTGCCGCCCGCTCTGGCCTCCG CGCCCAGGACCGGCTCATTGAGGTGAACGGGCAGAATGTGGAGGGACTGCGCCATGCTGAGGTGGTG CCAGCATCAAGGCACGGGAGGACGAGGCCCGGCTGCTGGTCGTGGACCCCGAGACAGATGAACACTTC AAGCGGCTTCGGGTCACACCCACCGAGGAGCACGTGGAAGGTCCTCTGCCGTCACCCGTCACCAATG GAACCAGCCCTGCCCAGCTCAATGGTGGCTCTGCGTGCTCGTCCCGAAGTGACCTGCCTGGTTCCGAC AAGGACACTGAGGATGGCAGTGCCTGGAAGCAAGATCCCTTCCAGGAGAGCGGCCTCCACCTGAGCCCC CACGGCGGCCGAGGCCAAGGAGAAGGCTCGAGCCATGCGAGTCAACAAGCGCGCGCCACAGATGGACT GGAACAGGAAGCGTGAAATCTTCAGCAACTTCTGAGCCCCTTCCTGCCTGTCTCGGGACCCTGGGAC CCCTCCCGCACGGACCTTGGGCCTCAGCCTGCCCCGAGCTCCCCAGCCTCAGTGGACTGGAGGGGTGG TCCTGCCATTGCCCAGAAATCAGCCCCAGCCCCGGTGAGCCCCCATCCTGCCCCTGCCCCACCAGGTAC TGGGGGCCTGTGGCAGCAAGATAGGGGAGAGAGACCCAGAGATGTGAGAGAGAGTCAGAGACAGAGA CAGAGAGAGAGAGAGAGAGACACAGAGAGAGAGAGAGCGAGCGAGCGCGCGGCAGCCGCG GGCGAGGGCCTTTGCTGCTCTGCCGGGGCCTGCTGACTGAAAGGAATTTGTGTTTTTGCTTTTTTTCC AAAAAGATCTCCAGCTCCACACATGTTTCCACTTAATAACCAGAGACCCCCCCCCTTCCCCTCCCCCT CCCCTCCCCCTTGGGACGCGCTCTAAATAATTGCAATAAAACAAAACCTTTCTCTGCAAACCATTTCCT CCCCGCCCCCTCCCCCTCAGCAGCGGCCGTCCTGAGTGGGAGTCCCTGGGACTTCCCAGTGGCCAAGT TGGGGCGCCCAGCCTCTTCGTGGGGACCTTGGGTAAGGCCAGGGAGGCCTGATGTGGCCGTAGGAGCT GCCCCTGCCCACCTGCCCTGGTGTGGGGGTCCCTAGGCCACACCCTGCTCCCACCCAGCTACCCTGT GCGCCTGTGCCCTGCTGGGGGCCTGGGCTCTCCGAGGGGCCTGAGGATGGAGGCCCCACGTCCCCGAG GAGGGCGGCCTCTGGACAGGCCCCTCATTCCGCGCGGCAGCTCCCAGGCCTGGGGAACGTAGGTGTG TGAGAGCGGCACCCGGGAAGGACGCCTGGCCTCTGGCTCAGCCCTGCTTGGCGGGCTCCCCCGTGGAC ACCCTGTTGACTTTGCACTTCCCTCCCGGGCCCGCACCCCCGAACCGACCACCGATCGACCGGCACC GCTGTTGCCTCGTAAGCCATAGCGCATGCGCGCTCTCAGGATAAAACAGGCCCTGCCTGGGA (SEQ ID NO: 3)

In some embodiments, SLC9A3 regulator 2 (SLC9A3R2) encodes the following amino acid sequence:
MAAPEPLRPRLCRLVRGEQGYGFHLHGEKGRRGQFIRRVEPGSPAEAAAALRAGDRLVEVNGVNVEGETHHQVVQRIKAVEGQTRLL VVDQETDEELRRRQLTCTEEMAQRGLPPAHDPWEPKPDWAHTGSHSSEAGKKDVSGPLRELRPRLCHLRKGPQGYGFNLHSDKSRP GQYIRSVDPGSPAARSGLRAQDRLIEVNGQNVEGLRHAEVVASIKAREDEARLLVVDPETDEHFKRLRVTPTEEHVEGPLPSPVTNGTSPAQLNGGSACSSRSDLPGSDKDTEDGSAWKQDPFQESGLHLSPTAAEAKEKARAMRVNKRAPQMDWNRKREIFSNF (SEQ ID NO: 4)

In some embodiments, the laminin subunit alpha 5 (LAMA5) fragment E8 cDNA has the following nucleic acid sequence:
GCTGCCGAGGATGCTGCTGGCCAGGCCCTGCAGCAGGCGGACCACACGTGGGCGACGGTGGTGCGGCAGGGCCTGGTGGACCGAGCCCAGCAGCTCCTGGC CAACAGCACTGCACTAGAAGAGGCCATGCTCCAGGAACAGCAGAGGCTGGGCCTTGTGTGGGCTGCCCTCCAGGGTGCCAGGACCCAGCTCCGAGATGTCC GGGCCAAGAAGGACCAGCTGGAGGCGCACATCCAGGCGGCGCAGGCCATGCTTGCCATGGACACAGACGAGACAAGCAAGAAGATCGCACATGCCAAGGCT GTGGCTGCTGAAGCCCAGGACACCGCCACCCGTGTGCAGTCCCAGCTGCAGGCCATGCAGGAGAATGTGGAGCGGTGGCAGGGCCAGTACGAGGGCCTGCGG GGCCAGGACCTGGGCCAGGCAGTGCTTGACGCAGGCCACTCAGTGTCCACCCTGGAGAAGACGCTGCCCAGCTGCTGGCCAAGCTGAGCATCCTGGAGAA CCGTGGGGTGCACAACGCCAGCCTGGCCCTGTCCGCCAGCATTGGCCGCGTGCGAGAGCTCATTGCCCAGGCCCGGGGGGCTGCCAGTAAGGTCAAGGTGCC CATGAAGTTCAACGGGCGCTCAGGGGTGCAGCTGCGGCACCCCACGGGATCTTGCCGACCTTGCTGCCTACACTGCCCTCAAGTTCTACCTGCAGGGCCCAG AGCCTGAGCCTGGGCAGGGTACCGAGGATCGCTTTGTGATGTACATGGGCAGCCGCCAGGCCACTGGGGACTACATGGGTGTGTCTCTGCGTGAC (SEQ ID NO: 5)

In some embodiments, the laminin subunit alpha 5 (LAMA5) fragment E8 encodes the following amino acid sequence:
AAEDAAGQALQQADHTWATVVRQGLVDRAQQLLANSTALEEAMLQEQQRLGLVWAALQGARTQLRDVR AKKDQLEAHIQAAQAMLAMDTDETSKKIAHAKAVAAEAQDTATRVQSQLQAMQENVERWQGQYEGLRGQ DLGQAVLDAGHSVSTLEKTLPQLLAKLSILENRGVHNASLALSASIGRVRELIAQARGAASKVKVPMKFNGRSGVQLRTPRDLADLAAYTALKFYLQGPEPEGQGTEDRFVMYMGSRQATGDYMGVSLR (SEQ ID NO: 6)

In some embodiments, the nucleic acid sequence is present in a non-viral vector. In some embodiments, the nucleic acid sequence is operably linked to an expression control sequence. In other embodiments, the nucleic acid is operably linked to two or more expression control sequences.

Various methods are known in the art and are suitable for introducing nucleic acids into cells, including viral and non-viral techniques. Typical non-viral techniques include, but are not limited to, electroporation, calcium phosphate-mediated introduction, nucleofection, sonoporation, heat shock, magnetofection, liposome-mediated introduction, microinjection, microprojectile-mediated introduction (nanoparticles), cationic polymer-mediated introduction (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.), or cell fusion.

In some embodiments, EVs containing the disclosed nucleic acid sequences are administered to cells in a subject, which can then induce the cells in the subject to become EV-producing cells. Accordingly, methods for reprogramming cells into EV-producing cells are also disclosed, which include exposing the cells to extracellular vesicles produced from cells containing or expressing the disclosed therapeutic genes.

Exosomes and microvesicles are distinct EVs based on their biogenesis and biophysical properties, including size and surface protein markers. Exosomes are small, homogeneous particles ranging in size from 40 to 150 nm, typically derived from the endocytic recycling pathway. During endocytosis, endocytic vesicles form at the plasma membrane and fuse to form early endosomes. These mature into late endosomes, where intraluminal vesicles become vesicle lumenal. Instead of fusing with lysosomes, these multivesicular bodies fuse directly with the plasma membrane, releasing exosomes into the extracellular space. Exosome biogenesis, protein cargo sorting, and release involve the endosomal sorting complex required for transport (ESCRT complex) and other associated proteins, such as Alix and Tsg101. In contrast, microvesicles are produced directly through the outward budding and fission of membrane vesicles from the plasma membrane; therefore, their surface markers are highly dependent on the composition of the membrane of origin. Furthermore, they tend to comprise a larger, more heterogeneous population of extracellular vesicles, ranging in diameter from 150 to 1000 nm. However, both types of vesicles have been shown to deliver functional mRNA, miRNA, and proteins to recipient cells.

In some embodiments, the polynucleotide is delivered intracellularly to cells via a gene gun, a microparticle or nanoparticle suitable for such delivery, transfection by electroporation, three-dimensional nanochannel electroporation, a tissue nanotransfection device, a liposome suitable for such delivery, or a deep local tissue nanoelectroinjection device. In some embodiments, a viral vector can be used. However, in other embodiments, the polynucleotide is not delivered by a virus.

Electroporation is a technique in which an electric field is applied to cells to increase the permeability of the cell membrane, allowing cargo (e.g., reprogramming factors) to be introduced into the cells. Electroporation is a common technique for introducing foreign DNA into cells.

Tissue nanotransfection enables the direct cytosolic delivery of cargo (e.g., reprogramming factors) into cells by applying a very strong and focused electric field through arrayed nanochannels that benignly nanoporate adjacent tissue cellular members and electrophoretically drive the cargo into the cells.

To express the polypeptide or functional nucleic acid, the nucleotide coding sequence can be inserted into an appropriate expression vector. Accordingly, also disclosed are non-viral vectors comprising a polynucleotide comprising a nucleic acid sequence disclosed herein, wherein the nucleic acid sequence is operably linked to an expression control sequence. In some embodiments, the nucleic acid sequence is operably linked to a single expression control sequence. In other embodiments, the nucleic acid sequence is operably linked to two or more separate expression control sequences.

Methods for constructing expression vectors containing gene sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, Plainview, N.Y., 1989) and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, N.Y., 1989).

Expression vectors generally contain regulatory elements necessary for the translation and/or transcription of an inserted coding sequence. For example, the coding sequence is preferably operably linked to a promoter and/or enhancer to help control the expression of the desired gene product.

Promoters used in biotechnology are of different types according to the intended type of control of gene expression. They can generally be divided into constitutive promoters, tissue- or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

Constitutive promoters direct expression in virtually all tissues and are largely, if not completely, independent of environmental and developmental factors. Because their expression is usually not conditioned by endogenous factors, constitutive promoters are usually active across species and even kingdoms. Examples of constitutive promoters include CMV, EF1a, SV40, PGK1, Ubc, human beta-actin, and CAG.

Tissue-specific or developmental stage-specific promoters direct the expression of a gene in a particular tissue(s) or at a particular developmental stage. In plants, promoter elements that express or affect the expression of genes in the vascular system, photosynthetic tissues, tubers, roots, and other vegetative organs, or seeds and other reproductive organs can be found in heterologous systems (e.g., distantly related species or even other kingdoms), but greatest specificity is generally achieved with homologous promoters (i.e., from the same species, genus, or family). This is likely because coordinate expression of transcription factors is required to regulate the promoter's activity.

The performance of inducible promoters is conditioned not by endogenous factors but by environmental conditions and external stimuli that can be artificially controlled. Within this group are promoters regulated by non-biological factors such as light, oxygen levels, high and low temperatures, and wounding. Because some of these factors are difficult to control outside of the experimental environment, promoters that respond to chemical compounds not naturally found in the organism of interest are of particular interest. Similarly, promoters that respond to antibiotics, copper, alcohols, steroids, and herbicides, among other compounds, have been adapted and refined to allow induction of gene activity at will and independently of other biological or non-biological factors.

The two most commonly used inducible expression systems for studying eukaryotic cell biology are termed Tet-off and Tet-on. The Tet-Off system utilizes the tetracycline transactivator (tTA) protein, created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in herpes simplex virus. The resulting tTA protein can bind to DNA at specific TetO operator sequences. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a minimal promoter, such as the CMV promoter. The entire set of several TetO sequences with a minimal promoter is called a tetracycline response element (TRE), because it responds to the binding of the tetracycline transactivator protein tTA by increasing the expression of the gene(s) downstream of that promoter. In the Tet-Off system, expression of TRE-controlled genes can be suppressed by tetracycline and its derivatives. They bind to tTA, rendering it unable to bind to the TRE sequence, thereby preventing transactivation of the TRE-controlled gene. The Tet-On system functions similarly but in the opposite manner. In the Tet-Off system, tTA can bind to the operator only when it is not bound to tetracycline or one of its derivatives, such as doxycycline, whereas in the Tet-On system, the rtTA protein can bind to the operator only when bound to tetracycline. Thus, introduction of doxycycline into the system initiates transcription of the gene product. The Tet-On system is sometimes preferred over the Tet-Off system due to its faster response.

In some embodiments, the nucleic acid sequences disclosed herein are operably linked to the same expression control sequence. Alternatively, internal ribosome entry site (IRES) elements can be used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at internal sites. IRES elements can be attached to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. The IRES element ensures that each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.

Disclosed are non-viral vectors containing one or more polynucleotides disclosed herein operably linked to an expression control sequence. Examples of such non-viral vectors include oligonucleotides, alone or in combination with suitable protein, polysaccharide, or lipid formulations. Non-viral methods offer certain advantages over viral methods, simple large-scale production and low host immunogenicity being just two of them. Previously, low levels of transfection and gene expression were disadvantages of non-viral methods, but recent advances in vector technology have resulted in molecules and techniques with transfection efficiencies similar to those of viruses.

Examples of suitable non-viral vectors include, but are not limited to, pIRES-hrGFP-2a, pCMV6, pMAX, pCAG, pAd-IRES-GFP, and pCDNA3.0.

The disclosed compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that may be administered to a subject along with a nucleic acid or vector, without causing any undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition with which it comes into contact. The carrier will necessarily be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as will be known to one of skill in the art.

Treatment EV
Also disclosed are EVs produced ex vivo and incorporating a therapeutic cargo for use in treating FSHD. In some embodiments, the disclosed EVs can be any vesicles that can be secreted by cells. Cells secrete extracellular vesicles (EVs) with a wide range of diameters and functions, including apoptotic bodies (1-5 μm), microvesicles (100-1000 nm in size), and vesicles of endosomal origin known as exosomes (50-150 nm).

In some embodiments, the donor cells can be any donor cell capable of producing EVs, including (but not limited to) skin cells (e.g., fibroblasts, keratinocytes, skin stem cells), adipocytes, dendritic cells, peripheral blood mononuclear cells (PBMCs), pancreatic cells (e.g., ductal epithelial cells), liver cells (e.g., hepatocytes), and immune cells (e.g., T cells, macrophages, myeloid-derived suppressor cells).

The disclosed extracellular vesicles can be prepared by methods known in the art. For example, the disclosed extracellular vesicles can be prepared by expressing mRNA encoding a cell-targeting ligand in a eukaryotic cell. In some embodiments, the cell also expresses mRNA encoding a therapeutic cargo. The mRNA for the cell-targeting ligand and therapeutic cargo can be expressed from a vector that is transfected into a suitable production cell for producing the disclosed EVs. The mRNA for the cell-targeting ligand and therapeutic cargo can be expressed from the same vector (e.g., where the vector expresses the mRNA for the cell-targeting ligand and the therapeutic cargo from separate promoters), or the mRNA for the cell-targeting ligand and the therapeutic cargo can be expressed from separate vectors. The vector(s) for expressing the mRNA for the cell-targeting ligand and the therapeutic cargo can be packaged in a kit designed for preparing the disclosed extracellular vesicles.

Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.), ed. A. R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Additional carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

Pharmaceutical carriers are known to those of skill in the art. These are most typically standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those of skill in the art.

In addition to the molecule of choice, pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

Some of the present compositions may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl-, and arylamines, and substituted ethanolamines.

The compositions disclosed herein, including pharmaceutical compositions, can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, ophthalmically, intravaginally, rectally, intranasally, topically, etc., including topical intranasal administration or administration by inhalation.

The disclosed extracellular vesicles may incorporate therapeutic agents, which deliver the agents to muscle cells. Suitable therapeutic agents include, but are not limited to, therapeutic agents (e.g., small molecule drugs), therapeutic proteins, and therapeutic nucleic acids (e.g., therapeutic RNA). In some embodiments, the disclosed extracellular vesicles contain therapeutic RNA (also referred to herein as "cargo RNA"). In certain embodiments, the cargo is DUX4 RNAi.

For example, in some embodiments, the cell targeting protein also includes an RNA domain (e.g., at the cytoplasmic C-terminus of the fusion protein) that binds to one or more RNA motifs present in the cargo RNA to package the cargo RNA into extracellular vesicles before the extracellular vesicles are secreted from the cell. As such, the protein can function as both a "cell targeting protein" and a "packaging protein." In some embodiments, the packaging protein can be referred to as an extracellular vesicle integrating protein or an "EV integrating protein."

The cargo RNA of the disclosed extracellular vesicles can be of any suitable length. For example, in some embodiments, the cargo RNA can have a nucleotide length of at least about 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 100 nt, 200 nt, 500 nt, 1000 nt, 2000 nt, 5000 nt, or more. In other embodiments, the cargo RNA can have a nucleotide length of about 5000 nt, 2000 nt, 1000 nt, 500 nt, 200 nt, 100 nt, 50 nt, 40 nt, 30 nt, 20 nt, or 10 nt or less. In still further embodiments, the cargo RNA can have a nucleotide length within these contemplated nucleotide length ranges, e.g., a range of about 10 nt to 5000 nt, or other ranges of nucleotide lengths. The cargo RNA of the disclosed extracellular vesicles may be relatively long, for example, the cargo RNA comprises mRNA or another relatively long RNA.

In some embodiments, the therapeutic cargo is a membrane-permeable pharmacological compound that is secreted by the cell and then incorporated into the EV.

Transfection-based approaches have been proposed to achieve small RNA incorporation into EVs. Other reports have shown that vector-induced expression of small RNAs within cells can be used to achieve small RNA incorporation into EVs. Alternatively, EV donor cells may be directly transfected with small RNAs. Incubating tumor cells with chemotherapeutic drugs is another method for packaging drugs into EVs. To stimulate the formation of drug-loaded EVs, cells are irradiated with ultraviolet light to induce apoptosis. Alternative approaches, such as fusogenic liposomes, also result in drug incorporation into EVs.

In some embodiments, the therapeutic cargo is loaded into the EVs by diffusion through a concentration gradient.

Methods Disclosed herein are methods for delivering diagnostic or therapeutic cargo to muscle cells using the disclosed EVs. Accordingly, also disclosed herein are methods for treating any disease or condition associated with muscle cells. For example, the disclosed EVs can be used to treat facioscapulohumeral muscular dystrophy (FSHD).

The disclosed EVs may be administered to a subject by any suitable means. Administration to a human or animal subject may be selected from parenteral, intramuscular, intracerebral, intravascular, subcutaneous, or transdermal administration. Typically, the delivery method is by injection. Preferably, the injection is intramuscular or intravascular (e.g., intravenous). A physician will be able to determine the route of administration required for each particular patient.

EV is preferably delivered as a composition. The composition may be formulated for parenteral, intramuscular, intracerebral, intravascular (including intravenous), subcutaneous, or transdermal administration. Compositions for parenteral administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives. EV may be formulated in pharmaceutical compositions that may include, in addition to EV, pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and other pharmaceutically acceptable carriers or excipients.

Parenteral administration is generally characterized by injection, such as subcutaneous, intramuscular, or intravenous. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready for combination with a solvent immediately prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready for combination with a vehicle immediately prior to use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

For intravenous administration, suitable carriers include saline or phosphate-buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, as well as mixtures thereof. Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Bacteriostatic or fungistatic concentrations of antibacterial agents, including phenol or cresol, mercuric, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride, must be added to parenteral formulations packaged in multidose containers. Isotonicity agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment. The concentration of the pharmaceutically active compound is adjusted so that an effective amount is provided upon injection to produce the desired pharmacological effect. The exact dose depends on the age, weight, and condition of the patient or animal, as is known in the art.

Unit-dose parenteral preparations may be packaged in an ampule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile, as known and practiced in the art.

A therapeutically effective amount of the composition is administered. The dosage can be determined according to various parameters, particularly the severity of the patient's condition, age, and weight, the route of administration, and the required regimen. A physician will be able to determine the route of administration and dosage required for any particular patient. Optimal dosages may vary depending on the relative potency of individual constructs and can generally be estimated based on the EC50 found to be effective in in vitro and in vivo animal models. Generally, dosages are 0.01 mg to 100 mg/kg of body weight. A typical daily dose is about 0.1 to 50 mg/kg of body weight, preferably about 0.1 mg/kg to 10 mg/kg, depending on the potency of the particular construct, the age, weight, and condition of the subject being treated, the severity of the disease, and the frequency and route of administration. Different dosages of the construct can be administered depending on whether administration is by intramuscular or systemic (intravenous or subcutaneous) injection.

Preferably, the dose for a single intramuscular injection is in the range of approximately 5-20 μg. Preferably, the dose for a single or multiple systemic injection is in the range of 10-100 mg/kg of body weight.

Due to clearance of the construct (and degradation of any targeted molecules), the patient may need to be treated repeatedly, for example, one or more times daily, weekly, monthly, or yearly. One of skill in the art can readily estimate dosing repetition rates based on measured residence time and concentration of the construct in bodily fluids or tissues. After successful treatment, it may be desirable to subject the patient to maintenance therapy, with the construct administered at a maintenance dose ranging from 0.01 mg to 100 mg/kg of body weight, once or more per day to once every 20 years.

Specific Embodiments Embodiment 1. A composition comprising extracellular vesicles (EVs) produced from donor somatic cells modified to express NHERF1, NHERF2, a fusion protein comprising an E8 fragment of laminin and an exosomal or lysosomal transmembrane protein, or a combination thereof.

Embodiment 1. The embodiment of claim 1, wherein the donor cells are autologous or allogeneic.

Embodiment 3. The composition of embodiment 1 or 2, wherein the donor cells are skin cells or muscle cells.

Embodiment 4. The composition of any one of embodiments 1 to 3, wherein the EV encapsulates a therapeutic cargo.

Embodiment 5. The composition of embodiment 4, wherein the therapeutic cargo comprises a DUX4 silencing oligonucleotide or a nucleic acid encoding a DUX4 silencing oligonucleotide.

Embodiment 6. A method of treating facioscapulohumeral muscular dystrophy (FSHD) in a subject, comprising administering to the subject an effective amount of the composition described in any one of embodiments 1 to 5.

Embodiment 7. A method for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject, comprising intracellular delivery into skin cells of the subject of a polynucleotide comprising a nucleic acid sequence encoding NHERF1, NHERF2, a fusion protein comprising an E8 fragment of laminin and an exosomal or lysosomal transmembrane protein, or a combination thereof, and a nucleic acid sequence encoding a DUX4 silencing oligonucleotide.

Several embodiments of the present invention have been described. Nevertheless, it should be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Example 1: P38β modified EV as a therapeutic strategy for muscular dystrophy
Mitogen-activated protein kinases (MAPKs) are a type of protein kinase that are thought to be involved in cell signaling processes such as proliferation, differentiation, migration, and apoptosis. The p38 MAPK family consists of four kinases: p38α, p38β, p38γ, and p38δ. P38β is highly expressed in brain and muscle. p38 MAPK plays an important role in muscle differentiation and myogenesis by activating myogenic regulatory factors (MRFs). In recent years, p38α/P38β have attracted attention for their role in several muscular dystrophies by increasing inflammation and muscle degeneration.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal expression of DUX4 and is characterized by progressive skeletal muscle weakness and atrophy. During early embryonic development, the transcription factor double homeobox 4 (DUX4) is highly expressed and induces the expression of genes involved in pre- and post-implantation development. However, DUX4 expression is suppressed in adult tissues. Recent studies have demonstrated that small interfering RNA-mediated knockdown of p38α/p38β reduces DUX4 mRNA expression in vivo and in vitro without inhibiting muscle differentiation. Furthermore, p38α/p38β inhibition reduced catabolic effects and muscle atrophy induced by activin A activation in mice. The P38β isoform has been identified as a key mediator of muscle protein degradation by activating autophagy and the ubiquitin proteasome pathway (UPP), and may be associated with cancer-induced muscle atrophy and cancer cachexia in patients. Additionally, cachexia is a wasting syndrome associated with muscle mass loss in several chronic diseases, including diabetes, cancer, chronic obstructive pulmonary disease, and chronic kidney disease (CKD). Targeting P38β may represent a novel therapeutic approach for treating muscular dystrophies, such as FSHD and cachectic wasting syndrome.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. References cited herein and the materials for which they are cited are specifically incorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

References 1. E. Mocciaro, et al, Cells, 2021, 10.
2. J. Oliva, S. et al, J Pharmacol Exp Ther, 2019, 370, 219-230.
3. H. Ding, et al, J Cachexia Sarcopenia Muscle, 2017, 8, 202-212.
4. Z. Liu, et al, Cell Stress, 2018, 2, 311-324.
5. T. Yoshida, et al, Am J Med Sci, 2015, 350, 250-256.

Claims (7)

A composition comprising extracellular vesicles (EVs) produced from donor somatic cells modified to express NHERF1, NHERF2, a fusion protein comprising an E8 fragment of laminin and an exosomal or lysosomal transmembrane protein, or a combination thereof. The composition of claim 1, wherein the donor cells are autologous or allogeneic. The composition of claim 1, wherein the donor cells are skin cells or muscle cells. The composition of claim 1, wherein the EV encapsulates a therapeutic cargo. The composition of claim 4, wherein the therapeutic cargo comprises a DUX4 silencing oligonucleotide or a nucleic acid encoding a DUX4 silencing oligonucleotide. A method for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject, comprising administering to the subject an effective amount of the composition of claim 1. A method for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject, comprising intracellular delivery of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising NHERF1, NHERF2, an E8 fragment of laminin and an exosomal or lysosomal transmembrane protein, or a combination thereof, and a nucleic acid sequence encoding a DUX4 silencing oligonucleotide into skin cells of the subject.