EP4615418A1 - Designer extracellular vesicles for targeted delivery to muscle cells - Google Patents

Abstract

Disclosed herein are designer extracellular vesicles (EVs) that target muscle cells. For example, in some embodiments, the EVs are decorated NHERF1, NHERF2, a fusion protein containing an E8 fragment of laminin and an exosomal or lysosomal transmembrane protein, or a combination thereof. These EVs can in some embodiments, be used to deliver diagnostic and/or therapeutic cargo to muscle cells in a subject in need thereof. In some embodiments, these EVs are loaded with 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.

Description

DESIGNER EXTRACELLULAR VESICLES FOR TARGETED DELIVERY TO MUSCLE CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 63/383,188, filed November 10, 2022, which is hereby incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a sequence listing filed in ST.26 format entitled “321501-2660 Sequence Listing” created on August 24, 2023, having 11,940 bytes. The content of the sequence listing is incorporated herein in its entirety.

BACKGROUND

Facioscapulohumeral muscular dystrophy (FSHD) is a rare, progressive and disabling disease for which there are no approved treatments. The disease is characterized by progressive skeletal muscle loss that initially causes weakness in muscles in the face, shoulders, arms and trunk, and progresses to weakness throughout the lower body. Skeletal muscle weakness results in significant physical limitations, including an inability to smile and difficulty using arms for activities, with many patients ultimately becoming dependent upon the use of a wheelchair for daily mobility.

FSHD is caused by aberrant expression of DUX4 in skeletal muscle, resulting in the inappropriate presence of DUX4 protein. Normally, DUX4 gene expression is limited to early embryonic development, after which time the DUX4 gene is silenced. In patients with FSHD, the DUX4 gene is unsilenced as a result of a genetic mutation. The result is death of muscle and its replacement by fat, resulting in skeletal muscle weakness and progressive disability.

SUMMARY

Disclosed herein are designer extracellular vesicles (EVs) that target skeletal muscle cells and precursor muscle cells, such as myoblasts and satellite cells, engineered to express NHERF1 , NHERF2, a fusion protein containing an E8 fragment of laminin and an exosomal or lysosomal transmembrane protein. For example, in some embodiments, the designer EVs are derived from somatic cells genetically engineered to express NHERF1 , or NHERF2, or the 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 support cells (e.g., satellite cells, myoblasts), engineered to express NHERF1 , or NHERF2, or the E8 fragment of laminin associated with an exosomal or lysosomal transmembrane protein, which is expected to enhance their intrinsic tropism toward muscle tissue. In some embodiments, the method involves functionalizing EVs isolated from somatic cells, including muscle cells and their respective precursor support cells, with NHERF1 , or NHERF2, or the E8 fragment of laminin associated to an exosomal or lysosomal protein. NHERF1 and/or NHERF2 will target the CD34 receptor in myogenic tissue, while the E8 fragment of laminin will target the oc7p1 -integrin in sarcolemma.

These EVs can in some embodiments, be used to deliver diagnostic and/or therapeutic cargo to muscle cells in a subject in need thereof.

Therefore, also disclosed herein is a method for treating any disease or condition associated with muscle cells such as Duchenne muscular dystrophy (DMD), Limb-girdle muscular dystrophy 1C (LGMD1C), Facioscapulohumeral Muscular Dystrophy (FSHMD), Becker muscular dystrophy (BMD), Amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, Myasthenia gravis, Myopathy, among others. Therefore, the disclosed EVs can be used to treat one or more of these pathologies.

In some embodiments, these EVs are loaded with DUX4 RNAi, p38a, p38|3 (Mapk14) which also suppresses DUX4 expression, myostatin, NF-KB/p65, and can therefore be used to treat a disease in a subject. Therefore, also disclosed herein are methods of treating FSHD, DMD, and LGMD1C in a subject that involves engineering the cells of the subject to produce therapeutic EVs that target and deliver therapeutic cargo to muscle cells.

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

DESCRIPTION OF DRAWINGS

FIGs. 1A-1G show isolation and characterization of shRNA p38b loaded engineered EVs. FIG. 1A) Representative immunofluorescence images of donor cells, in this case primary mouse embryonic fibroblast (PMEFs), showing positive expression of the GFP reported included in the expression plasmid used for EV engineering at 24 hours after electroporation, FIG. 1 B) qRT-PCR results showing robust GFP upregulation in donor cells at 24 at 24 hours after electroporation, and FIG. 1C) PCR product showing positive expression of the lentiviral plasmid in scramble (control) and shRNA p38b transfected donor cells. FIG. 1 D) Transcripts gene level expression of GFP confirming effective loading of the shRNA p38b in the engineered EVs. Nanotracking particle analysis showing FIG. 1E) particle concentration and FIG. 1F) size distribution for shRNA p38b loaded engineered EVs, sham EVs, and naive EVs (released from nontransfected donor cells). FIG. 1G) PCR product showing the positive expression of the lentiviral backbone in scramble and shRNA p38b loaded engineered EVs. (One way- Anova *p<0.05 n=3).

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, 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, since the scope of the present disclosure will be limited only by the appended claims.

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

Unless defined otherwise, 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 the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for 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 virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

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

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

It must be noted that, as used in the 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. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature 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, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological 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 ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as 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 of reduction in between as compared to native or control levels.

The term “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid sidechains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have 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 a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein 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, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, 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.

The phrase “nucleic acid” as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids can also include nucleotide analogs (e.g., Brdll), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

A “nucleotide” as used herein is a molecule that contains a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The term “oligonucleotide” is sometimes used to refer to a molecule that contains two or more nucleotides linked together. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1 -yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example 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 moieties. Modifications to nucleotides are well known in the art and would include, for example, 5-methylcytosine (5-me-C), 5 hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.

Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the 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 has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). “Plasmid” and “vector” are used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.

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

For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

100 times the fraction W/Z, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, 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 achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.

By “specifically hybridizes” is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a c-met nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

The term “stringent hybridization conditions” as used herein mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5X SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt’s solution, 10% dextran sulfate, and 20 pg/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 wash 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. The “control elements” or “regulatory sequences” are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.

A “promoter” is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard 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 within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers, like promoters, also often contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.

An “endogenous” enhancer/promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e. , molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.

Muscle Cell Targeting Extracellular vehicles (EVs)

Disclosed herein are EVs that target muscle cells that can be loaded with therapeutic and/or diagnostic cargo. In some embodiments, the method involves collection of muscle cells from the subject and isolation of EVs that enhance tropism for muscle tissue. In other embodiments, the method involves engineering cells of the subject to produce therapeutic EVs. In some embodiments, the method involves collecting EVs produced ex vivo and loading them with therapeutic cargo. Finally, in some embodiments, the method includes the post-synthesis functionalization of the EVs.

Engineering Patient Cells to Produce Therapeutic EVs

Disclosed are methods of reprogramming cells of the subject into EV-producing cells that involve delivering intracellularly into cells a polynucleotide comprising nucleic acid sequences encoding muscle cells ligands and optionally a therapeutic cargo. In some embodiments, the cells can be any cell in the subject able to produce EVs, including (but not limited to) skin cells (e.g., fibroblasts, keratinocytes, skin stem cells), adipocytes, dendritic cells, peripheral blood mononuclear cells (PBMC), pancreatic cells (e.g., ductal epithelial cells), liver cells (e.g., hepatocytes), immune cells (e.g., T cells, macrophages, myeloid derived suppressor cells).

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

In some embodiments, a SLC9A3 regulator 1 (SLC9A3R1) cDNA has the nucleic acid sequence: AGACGCCGCGCGGGGCGGGGATTGGTCTGTGGTCCTCTCTCGGCTCCTCGCGGC TCGCGGCGGCCGACGGTTCCTGGGACACCTGCTTGCTTGGCCCGTCCGGCGGCT CAGGGCTTCTCTGCTGCGCTCCCGGTTCGCTGGACGGGAAGAAGGGCTGGGCCG TCCCGTCCCGTCCCCATCGGAACCCCAAGTCGCGCCGCTGACCCGTCGCAGGGC GAGATGAGCGCGGACGCAGCGGCCGGGGCGCCCCTGCCCCGGCTCTGCTGCCT GGAGAAGGGTCCGAACGGCTACGGCTTCCACCTGCACGGGGAGAAGGGCAAGTT GGGCCAGTACATCCGGCTGGTGGAGCCCGGCTCGCCGGCCGAGAAGGCGGGGC TGCTGGCGGGGGACCGGCTGGTGGAGGTGAACGGCGAAAACGTGGAGAAGGAGA CCCACCAGCAGGTGGTGAGCCGCATCCGCGCCGCACTCAACGCCGTGCGCCTGC TGGTGGTCGACCCCGAGACGGACGAGCAGCTGCAGAAGCTCGGCGTCCAGGTCC GAGAGGAGCTGCTGCGCGCCCAGGAAGCGCCGGGGCAGGCCGAGCCGCCGGCC GCCGCCGAGGTGCAGGGGGCTGGCAACGAAAATGAGCCTCGCGAGGCCGACAAG AGCCACCCGGAGCAGCGCGAGCTTCGGCCTCGGCTCTGTACCATGAAGAAGGGC CCCAGTGGCTATGGCTTCAACCTGCACAGCGACAAGTCCAAGCCAGGCCAGTTCA TCCGGTCAGTGGACCCAGACTCCCCGGCTGAGGCTTCAGGGCTCCGGGCCCAGG ATCGCATTGTGGAGGTGAACGGGGTCTGCATGGAGGGGAAGCAGCATGGGGACG TGGTGTCCGCCATCAGGGCTGGCGGGGACGAGACCAAGCTGCTGGTGGTGGACA GGGAAACTGACGAGTTCTTCAAGAAATGCAGAGTGATCCCATCTCAGGAGCACCTG AATGGTCCCCTGCCTGTGCCCTTCACCAATGGGGAGATACAGAAGGAGAACAGTC GTGAAGCCCTGGCAGAGGCAGCCTTGGAGAGCCCCAGGCCAGCCCTGGTGAGAT CCGCCTCCAGTGACACCAGCGAGGAGCTGAATTCCCAAGACAGCCCCCCAAAACA GGACTCCACAGCGCCCTCGTCTACCTCCTCCTCCGACCCCATCCTAGACTTCAACA TCTCCCTGGCCATGGCCAAAGAGAGGGCCCACCAGAAACGCAGCAGCAAACGGG CCCCGCAGATGGACTGGAGCAAGAAAAACGAACTCTTCAGCAACCTCTGAGCGCC CTGCTGCCACCCAGTGACTGGCAGGGCCGAGCCAGCATTCCACCCCACCTTTTTC CTTCTCCCCAATTACTCCCCTGAATCAATGTACAAATCAGCACCCACATCCCCTTTC TTGACAAATGATTTTTCTAGAGAACTATGTTCTTCCCTGACTTTAGGGAAGGTGAAT GTGTTCCCGTCCTCCCGCAGTCAGAAAGGAGACTCTGCCTCCCTCCTCCTCACTGA GTGCCTCATCCTACCGGGTGTCCCTTTGCCACCCTGCCTGGGACATCGCTGGAAC CTGCACCATGCCAGGATCATGGGACCAGGCGAGAGGGCACCCTCCCTTCCTCCCC CATGTGATAAATGGGTCCAGGGCTGATCAAAGAACTCTGACTGCAGAACTGCCGCT CTCAGTGGACAGGGCATCTGTTACCCTGAGACCTGTGGCAGACACGTCTTGTTTTC ATTTGATTTTTGTTAAGAGTGCAGTATTGCAGAGTCTAGAGGAATTTTTGTTTCCTTG ATTAACATGATTTTCCTGGTTGTTACATCCAGGGCATGGCAGTGGCCTCAGCCTTAA ACTTTTGTTCCTACTCCCACCCTCAGCGAACTGGGCAGCACGGGGAGGGTTTGGC TACCCCTGCCCATCCCTGAGCCAGGTACCACCATTGTAAGGAAACACTTTCAGAAA TTCAGCTGGTTCCTCCAAA (SEQ ID N0:1).

In some embodiments, a SLC9A3 regulator 1 (SLC9A3R1) mRNA encodes the amino acid sequence MSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKGKLGQYIRLVEPGSPAEKAGLLAGD RLVEVNGENVEKETHQQVVSRIRAALNAVRLLVVDPETDEQLQKLGVQVREELLRAQE APGQAEPPAAAEVQGAGNENEPREADKSHPEQRELRPRLCTMKKGPSGYGFNLHSD KSKPGQFIRSVDPDSPAEASGLRAQDRIVEVNGVCMEGKQHGDVVSAIRAGGDETKLL VVDRETDEFFKKCRVIPSQEHLNGPLPVPFTNGEIQKENSREALAEAALESPRPALVRS ASSDTSEELNSQDSPPKQDSTAPSSTSSSDPILDFNISLAMAKERAHQKRSSKRAPQM DWSKKNELFSNL (SEQ ID NO:2).

In some embodiments, a SLC9A3 regulator 2 (SLC9A3R2) cDNA has the nucleic acid sequence: GAACAGGAGCCGCCGCTGAAGCCACCGCCGGGTGCCCAGCGCCGCCGCCGCCC CCGAGCTCCCCCGCGCCCCTGCCCGCGGGCGGCCGGTGGGCAGCGGGCGCCAT GGCCGCGCCGGAGCCGCTGCGGCCGCGCCTGTGCCGCTTGGTGCGCGGAGAGC AGGGCTACGGCTTCCACCTGCACGGCGAGAAGGGCCGCCGCGGGCAGTTCATCC GGCGCGTGGAACCCGGTTCCCCCGCCGAGGCCGCCGCGCTGCGCGCTGGGGAC CGCCTGGTCGAGGTCAACGGCGTCAACGTGGAGGGCGAGACGCACCACCAGGTG GTGCAAAGGATCAAGGCTGTGGAGGGGCAGACTCGGCTGCTGGTGGTGGACCAG GAGACAGATGAGGAGCTCCGCCGGCGGCAGCTGACCTGTACCGAGGAGATGGCC CAGCGAGGGCTCCCACCCGCCCACGACCCCTGGGAGCCGAAGCCAGACTGGGCA CACACCGGCAGCCACAGCTCCGAAGCTGGCAAGAAGGATGTCAGTGGGCCCCTGA GGGAGCTGCGCCCTCGGCTCTGCCACCTGCGAAAGGGACCTCAGGGCTATGGGT

TCAACCTGCATAGTGACAAGTCCCGGCCCGGCCAGTACATCCGCTCTGTGGACCC

GGGCTCACCTGCCGCCCGCTCTGGCCTCCGCGCCCAGGACCGGCTCATTGAGGT

GAACGGGCAGAATGTGGAGGGACTGCGCCATGCTGAGGTGGTGGCCAGCATCAA

GGCACGGGAGGACGAGGCCCGGCTGCTGGTCGTGGACCCCGAGACAGATGAACA

CTTCAAGCGGCTTCGGGTCACACCCACCGAGGAGCACGTGGAAGGTCCTCTGCCG

TCACCCGTCACCAATGGAACCAGCCCTGCCCAGCTCAATGGTGGCTCTGCGTGCT

CGTCCCGAAGTGACCTGCCTGGTTCCGACAAGGACACTGAGGATGGCAGTGCCTG

GAAGCAAGATCCCTTCCAGGAGAGCGGCCTCCACCTGAGCCCCACGGCGGCCGA

GGCCAAGGAGAAGGCTCGAGCCATGCGAGTCAACAAGCGCGCGCCACAGATGGA

CTGGAACAGGAAGCGTGAAATCTTCAGCAACTTCTGAGCCCCTTCCTGCCTGTCTC

GGGACCCTGGGACCCCTCCCGCACGGACCTTGGGCCTCAGCCTGCCCCGAGCTC

CCCCAGCCTCAGTGGACTGGAGGGTGGTCCTGCCATTGCCCAGAAATCAGCCCCA

GCCCCGGTGAGCCCCCATCCTGCCCCTGCCCACCAGGTACTGGGGGCCTGTGGC

AGCAAGATAGGGGGAGAGAGACCCAGAGATGTGAGAGAGAGTCAGAGACAGAGA

CAGAGAGAGAGAGAGAGAGACACAGAGAGAGACAGAGAGAGAGCGAGCGAGCGC

GCGGCAGCCGCGGGGCGAGGGCCTTTGCTGCTCTGCCGGGGCCTGCTGACTGAA

AGGAATTTGTGTTTTTGCTTTTTTTCCAAAAAGATCTCCAGCTCCACACATGTTTCCA

CTTAATACCAGAGACCCCCCCCCTTCCCCTCCCCCTTCCCCTCCCCCTTGGGACGC

GCTCTAAATAATTGCAATAAAACAAACCTTTCTCTGCAAACCATTTCCTCCCCGCCC

CCTCCCCTCAGCAGCGGCCGTCCTGAGTGGGAGTCCCTGGGACTTCCCAGTGGCC

AAGTTGGGGCGCCCAGCCTCTTCGTGGGGACCTTGGGTAAGGCCAGGGAGGCCT

GATGTGGCCGTAGGAGCTGCCCCTGCCCACCTGCCCTGGTGTGGGGGTCCCTAG

GCCACACCCTGCTCCCCACCCAGCTACCCTGTGCGCCTGTGCCCTGCTGGGGGCC

TGGGCTCTCCGAGGGGCCTGAGGATGGAGGCCCCACGTCCCCGAGGAGGGCGG

CCTCTGGACAGGCCCCTCATTCCGCGCGGCAGCTCCCAGGCCTGGGGAACGTAG

GTGTGTGAGAGCGGCACCCGGGAAGGACGCCTGGCCTCTGGCTCAGCCCTGCTT GGCGGGCTCCCCCGTGGACACCCTGTTGACTTTGCACTTCCCTCCCGGGCCCCGC

ACCCCCGAACCGACCACCGATCGACCGGCACCGCTGTTGCCTCGTAAGCCATAGC GCATGCGCGCTCTCAGGATAAACAGGCCCTGCCTGGGA (SEQ ID N0:3).

In some embodiments, a SLC9A3 regulator 2 (SLC9A3R2) encodes the amino acid sequence:

MAAPEPLRPRLCRLVRGEQGYGFHLHGEKGRRGQFIRRVEPGSPAEAAALRAGDRLV

EVNGVNVEGETHHQVVQRIKAVEGQTRLLVVDQETDEELRRRQLTCTEEMAQRGLPP AHDPWEPKPDWAHTGSHSSEAGKKDVSGPLRELRPRLCHLRKGPQGYGFNLHSDKS RPGQYIRSVDPGSPAARSGLRAQDRLIEVNGQNVEGLRHAEVVASIKAREDEARLLVV DPETDEHFKRLRVTPTEEHVEGPLPSPVTNGTSPAQLNGGSACSSRSDLPGSDKDTE DGSAWKQDPFQESGLHLSPTAAEAKEKARAMRVNKRAPQMDWNRKREIFSNF (SEQ ID N0:4).

In some embodiments, a laminin subunit alpha 5 (LAMA5) fragment E8 cDNA has the nucleic acid sequence: GCTGCCGAGGATGCTGCTGGCCAGGCCCTGCAGCAGGCGGACCACACGTGGGCG ACGGTGGTGCGGCAGGGCCTGGTGGACCGAGCCCAGCAGCTCCTGGCCAACAGC ACTGCACTAGAAGAGGCCATGCTCCAGGAACAGCAGAGGCTGGGCCTTGTGTGGG CTGCCCTCCAGGGTGCCAGGACCCAGCTCCGAGATGTCCGGGCCAAGAAGGACC AGCTGGAGGCGCACATCCAGGCGGCGCAGGCCATGCTTGCCATGGACACAGACG AGACAAGCAAGAAGATCGCACATGCCAAGGCTGTGGCTGCTGAAGCCCAGGACAC CGCCACCCGTGTGCAGTCCCAGCTGCAGGCCATGCAGGAGAATGTGGAGCGGTG GCAGGGCCAGTACGAGGGCCTGCGGGGCCAGGACCTGGGCCAGGCAGTGCTTGA CGCAGGCCACTCAGTGTCCACCCTGGAGAAGACGCTGCCCCAGCTGCTGGCCAAG CTGAGCATCCTGGAGAACCGTGGGGTGCACAACGCCAGCCTGGCCCTGTCCGCCA GCATTGGCCGCGTGCGAGAGCTCATTGCCCAGGCCCGGGGGGCTGCCAGTAAGG TCAAGGTGCCCATGAAGTTCAACGGGCGCTCAGGGGTGCAGCTGCGCACCCCACG GGATCTTGCCGACCTTGCTGCCTACACTGCCCTCAAGTTCTACCTGCAGGGCCCAG AGCCTGAGCCTGGGCAGGGTACCGAGGATCGCTTTGTGATGTACATGGGCAGCCG CCAGGCCACTGGGGACTACATGGGTGTGTCTCTGCGTGAC (SEQ ID NO:5).

In some embodiments, a laminin subunit alpha 5 (I.AMA5) fragment E8 encodes the amino acid sequence: AAEDAAGQALQQADHTWATVVRQGLVDRAQQLLANSTALEEAMLQEQQRLGLVWAA LQGARTQLRDVRAKKDQLEAHIQAAQAMLAMDTDETSKKIAHAKAVAAEAQDTATRVQ SQLQAMQENVERWQGQYEGLRGQDLGQAVLDAGHSVSTLEKTLPQLLAKLSILENRG VHNASLALSASIGRVRELIAQARGAASKVKVPMKFNGRSGVQLRTPRDLADLAAYTALK FYLQGPEPEPGQGTEDRFVMYMGSRQATGDYMGVSLR (SEQ ID NO:6).

In some embodiments, the nucleic acid sequences are present in non-viral vectors. In some embodiments, the nucleic acid sequences are operably linked to an expression control sequence. In other embodiments the nucleic acids are operably linked to two or more expression control sequences. A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.

In some embodiments, EVs containing the disclosed nucleic acid sequences are administered to the cells of the subject, which can then induce cells in the subject to be EV-producing cells. Therefore, also disclosed is a method of reprogramming cells into EV-producing cells that involves exposing the cell with an extracellular vesicle produced from a cell containing or expressing the disclosed therapeutic genes.

Exosomes and microvesicles are EVs that differ based on their process of biogenesis and biophysical properties, including size and surface protein markers. Exosomes are homogenous small particles ranging from 40 to 150 nm in size and they are normally derived from the endocytic recycling pathway. In endocytosis, endocytic vesicles form at the plasma membrane and fuse to form early endosomes. These mature and become late endosomes where intraluminal vesicles bud off into an intra- vesicular lumen. Instead of fusing with the lysosome, these multivesicular bodies directly fuse with the plasma membrane and release 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, and hence, their surface markers are largely dependent on the composition of the membrane of origin. Further, they tend to constitute a larger and more heterogeneous population of extracellular vesicles, ranging from 150 to 1000 nm in diameter. However, both types of vesicles have been shown to deliver functional mRNA, miRNA and proteins to recipient cells.

In some embodiments, the polynucleotides are delivered to the cells intracellularly 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-topical tissue nanoelectroinjection device. In some embodiments, a viral vector can be used. However, in other embodiments, the polynucleotides are not delivered virally.

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

Tissue nanotransfection allows for direct cytosolic delivery of cargo (e.g., reprogramming factors) into cells by applying a highly intense and focused electric field through arrayed nanochannels, which benignly nanoporates the juxtaposing tissue cell members, and electrophoretically drives cargo into the cells.

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

Methods to construct expression vectors containing genetic 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 sequences necessary elements for the translation and/or transcription of the 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 be generally divided into constitutive promoters, tissue-specific or development-stage-specific promoters, inducible promoters, and synthetic promoters. Constitutive promoters direct expression in virtually all tissues and are largely, if not entirely, independent of environmental and developmental factors. As their expression is normally not conditioned by endogenous factors, constitutive promoters are usually active across species and even across kingdoms. Examples of constitutive promoters include CMV, EF1a, SV40, PGK1 , Ubc, Human beta actin, and CAG.

Tissue-specific or development-stage-specific promoters direct the expression of a gene in specific tissue(s) or at certain stages of development. For plants, promoter elements that are expressed 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 the most specificity is generally achieved with homologous promoters (i.e. from the same species, genus or family). This is probably because the coordinate expression of transcription factors is necessary for regulation of the promoter's activity.

The performance of inducible promoters is not conditioned to endogenous factors but to environmental conditions and external stimuli that can be artificially controlled. Within this group, there are promoters modulated by abiotic factors such as light, oxygen levels, heat, cold and wounding. Since some of these factors are difficult to control outside an experimental setting, promoters that respond to chemical compounds, not found naturally in the organism of interest, are of particular interest. Along those lines, promoters that respond to antibiotics, copper, alcohol, steroids, and herbicides, among other compounds, have been adapted and refined to allow the induction of gene activity at will and independently of other biotic or abiotic factors.

The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On. The Tet-Off system makes use of the tetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the Herpes Simplex Virus. The resulting tTA protein is able to 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 entirety of several TetO sequences with a minimal promoter is called a tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter. In a Tet-Off system, expression of TRE-controlled genes can be repressed by tetracycline and its derivatives. They bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE- controlled genes. A Tet-On system works similarly, but in the opposite fashion. While in a Tet-Off system, tTA is capable of binding the operator only if not bound to tetracycline or one of its derivatives, such as doxycycline, in a Tet-On system, the rtTA protein is capable of binding the operator only if bound by a tetracycline. Thus the introduction of doxycycline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred over Tet-Off for its faster responsiveness.

In some embodiments, the nucleic acid sequences disclosed herein are operably linked to the same expression control sequence. Alternatively, internal ribosome entry sites (IRES) elements can be used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, 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 the oligonucleotide alone or in combination with a suitable protein, polysaccharide or lipid formulation. Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded 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 compositions disclosed can be used therapeutically in combination with a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

Therapeutic EVs

Also disclosed are EVs produced ex vivo and loaded with therapeutic cargo for use in treating FSHD. The disclosed EVs can in some embodiments be any vesicle that can be secreted by a cell. Cells secrete extracellular vesicles (EVs) with a broad range of diameters and functions, including apoptotic bodies (1-5 pm), 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 able to produce EVs, including (but not limited to) skin cells (e.g., fibroblasts, keratinocytes, skin stem cells), adipocytes, dendritic cells, peripheral blood mononuclear cells (PBMC), pancreatic cells (e.g., ductal epithelial cells), liver cells (e.g., hepatocytes), immune cells (e.g., T cells, macrophages, myeloid derived suppressor cells).

The disclosed extracellular vesicles may be prepared by methods known in the art. For example, the disclosed extracellular vesicles may be prepared by expressing in a eukaryotic cell an mRNA that encodes the cell-targeting ligand. In some embodiments, the cell also expresses an mRNA that encodes a therapeutic cargo. The mRNA for the cell-targeting ligand and the therapeutic cargo may be expressed from vectors that are transfected into suitable production cells for producing the disclosed EVs. The mRNA for the cell-targeting ligand and the therapeutic cargo may 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 may be expressed from separate vectors. The vector or vectors for expressing the mRNA for the cell-targeting ligand and the therapeutic cargo may 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 the pharmaceutically-acceptable carrier 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, and more preferably from about 7 to about 7.5. Further 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 persons 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 skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous or nonaqueous 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, for example, antimicrobials, anti-oxidants, 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 compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, 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 an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

The herein disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeal ly, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant.

Therapeutic cargo

The disclosed extracellular vesicles may be loaded with a therapeutic agent, where the extracellular vesicles deliver the agent to muscle cells. Suitable therapeutic agents include but are not limited to therapeutic drugs (e.g., small molecule drugs), therapeutic proteins, and therapeutic nucleic acids (e.g., therapeutic RNA). In some embodiments, the disclosed extracellular vesicles comprise a therapeutic RNA (also referred to herein as a “cargo RNA”). In particular embodiments, the cargo is DUX4 RNAi.

For example, in some embodiments a cell-targeting protein also includes an RNA-domain (e.g., at a cytosolic C-terminus of the fusion protein) that binds to one or more RNA-motifs present in the cargo RNA in order to package the cargo RNA into the extracellular vesicle, prior to the extracellular vesicles being secreted from a cell. As such, the protein may function as both of a “cell-targeting protein” and a “packaging protein.” In some embodiments, the packaging protein may be referred to as extracellular vesicle-loading protein or “EV-loading protein.”

The cargo RNA of the disclosed extracellular vesicles may be of any suitable length. For example, in some embodiments the cargo RNA may 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 longer. In other embodiments, the cargo RNA may have a nucleotide length of no more than about 5000 nt, 2000 nt, 1000 nt, 500 nt, 200 nt, 100 TH Docket No. 321501-2660 nt, 50 nt, 40 nt, 30 nt, 20 nt, or 10 nt. In even further embodiments, the cargo RNA may have a nucleotide length within a range of these contemplated nucleotide lengths, for example, a nucleotide length between a range of about 10 nt-5000 nt, or other ranges. The cargo RNA of the disclosed extracellular vesicles may be relatively long, for example, where the cargo RNA comprises an mRNA or another relatively long RNA.

In some embodiments, the therapeutic cargo is a membrane-permeable pharmacological compound that is loaded into the EV after it is secreted by the cell.

To achieve loading of small RNAs into EVs, transfection-based approaches have been proposed. Other reports have shown that using vector- induced expression of small RNAs in cells, small RNA loading into EVs can be achieved. Alternatively, EV donor cells may be transfected with small RNAs directly. Incubation of tumor cells with chemotherapeutic drugs is also another method to package drugs into EVs. To stimulate formation of drug-loaded EVs, cells are irradiated with ultraviolet light to induce apoptosis. Alternative approaches such as fusogenic liposomes also leads loading drugs into EVs.

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

Methods

Disclosed herein are methods for delivering diagnostic or therapeutic cargo to muscle cells using the disclosed EVs. Therefore, also disclosed herein is a method 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 method of delivery is by injection. Preferably the injection is intramuscular or intravascular (e.g. intravenous). A physician will be able to determine the required route of administration for each particular patient.

The EVs are 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 which may also contain buffers, diluents and other suitable additives. The EVs may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, TH Docket No. 321501-2660 diluents, buffers, preservatives, and other pharmaceutically acceptable carriers or excipients and the like in addition to the EVs.

Parenteral administration is generally characterized by injection, such as subcutaneously, intramuscularly, or intravenously. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial 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, ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated ringers injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.

Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of 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 injection provides an effective amount to produce the desired pharmacological effect. The exact TH Docket No. 321501-2660 dose depends on the age, weight and condition of the patient or animal as is known in the art.

The unit-dose parenteral preparations can be packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile, as is known and practiced in the art.

A therapeutically effective amount of composition is administered. The dose may be determined according to various parameters, especially according to the severity of the condition, age, and weight of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. Optimum dosages may vary depending on the relative potency of individual constructs, and can generally be estimated based on EC50s found to be effective in vitro and in vivo animal models. In general, dosage is from 0.01 mg/kg to 100 mg per kg of body weight. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1 mg/kg to 10 mg/kg of body weight, according to the potency of the specific construct, the age, weight and condition of the subject to be treated, the severity of the disease and the frequency and route of administration. Different dosages of the construct may be administered depending on whether administration is by intramuscular injection or systemic (intravenous or subcutaneous) injection.

Preferably, the dose of a single intramuscular injection is in the range of about 5 to 20 pg. Preferably, the dose of single or multiple systemic injections is in the range of 10 to 100 mg/kg of body weight.

Due to construct clearance (and breakdown of any targeted molecule), the patient may have to be treated repeatedly, for example once or more daily, weekly, monthly or yearly. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the construct in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy, wherein the construct is administered in maintenance doses, ranging from 0.01 mg/kg to 100 mg per kg of body weight, once or more daily, to once every 20 years.

Specific Embodiments

Embodiment 1. A composition comprising extracellular vesicles (EVs) produced from donor somatic cells engineered to express NHERF1 , NHERF2, a fusion protein TH Docket No. 321501-2660 containing 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 embodiment w1 to 3, wherein the EVs encapsulate 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 a composition of any one of embodiments 1 to 5.

Embodiment ?. A method for treating Facioscapulohumeral muscular dystrophy (FSHD) in a subject, comprising delivering intracellularly into skin cells of the subject a polynucleotide comprising nucleic acid sequences encoding NHERF1 , NHERF2, a fusion protein containing 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.

A number of embodiments of the invention have been described. Nevertheless, it will 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.

EXAMPLES

Example 1: P38fi Engineered EVs as a Therapeutic Strategy for Muscular Dystrophy

Mitogen-activated protein kinases (MAPKs) are a type of protein kinase implicated in cell signaling processes such as growth, proliferation, migration, and apoptosis. The p38 MAPK family is composed of 4 kinases: p38a, P38|3, P38y, and p385. P38p presents a higher expression in the brain and muscle. p38 MAPKs play an important role in muscle differentiation and myogenesis by activation of myogenic regulatory factors (MRFs). Recently, p38a/P38|3 have gained a lot of attention for their TH Docket No. 321501-2660 role in several muscular dystrophies by increasing inflammation and muscle degeneration.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of DUX4 and FSHD is characterized by progressive skeletal muscle weakness and wasting. In the early stages of embryogenesis, the transcription factor double homeobox4 (DUX4) is highly expressed to induce the expression of genes involved in pre- and post-implantation. However, DUX4 expression is silent in the adult tissue. Recent studies have shown that small interfering RNA-mediated knockdown of p38a/P38p reduced the expression of DUX4 mRNA in-vivo and in vitro without preventing muscle differentiation. Moreover, the inhibition of p38a/ P38[3 reduced the catabolic effect and muscle atrophy of activin A-activated in mice. P38(3 isoform has been identified as a key mediator of muscle protein degradation by activation of autophagy and ubiquitin-proteasome pathways (UPP) which can be related with cancer- induced muscle wasting and cancer cachexia in patients. Additionally, cachexia is wasting syndrome associated with muscle mass loss in several chronic diseases such as diabetes, cancer, chronic obstructive pulmonary disease, and chronic kidney disease (CKD). P38b targeting may represent a novel therapeutical approach to treating muscular dystrophies such as FSHD and cachexia wasting syndrome.

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

WHAT IS CLAIMED IS:

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

2. The composition of claim 1 , wherein the donor cells are autologous or allogeneic.

3. The composition of claim 1 , wherein the donor cells are skin cells or muscle cells.

4. The composition of claim 1 , wherein the EVs encapsulate a therapeutic cargo.

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

6. A method of treating Facioscapulohumeral muscular dystrophy (FSHD) in a subject, comprising administering to the subject an effective amount of a composition of claim 1.

7. A method for treating Facioscapulohumeral muscular dystrophy (FSHD) in a subject, comprising delivering intracellularly into skin cells of the subject a polynucleotide comprising nucleic acid sequences encoding NHERF1, NHERF2, a fusion protein containing 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.