Micro RNA sponges useful for increasing MG29 expression and glucose uptake and treating metabolic disorders

Abstract

Provided herein are nucleic acids comprising at least one miRNA binding nucleotide sequence, vectors (e.g., AAV) comprising nucleic acids with at least one miRNA binding nucleotide sequence for inhibiting miRNAs that bind to a 3' UTR of an MG29 gene and methods of using the same to prevent or treat metabolic disorders, such as diabetes.

Description

MICRO RNA SPONGES USEFUL FOR INCREASING MG29 EXPRESSION AND GLUCOSE UPTAKE AND TREATING METABOLIC DISORDERS RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No.63 / 591,348, filed October 18, 2023, the entire contents of which are incorporated herein by reference. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (HNC0001PCT.xml; Size: 70,952 bytes; and Date of Creation: October 17, 2024) is herein incorporated by reference in its entirety. BACKGROUND

[0003] Type 2 diabetes (T2D), is a metabolic disorder characterized by hyperglycemia (high blood sugar) characterized by insulin resistance—the inability of the body cells to use insulin efficiently. In insulin resistance, muscle, fat, and liver cells do not respond properly to insulin and, as a result, increasing levels of insulin are needed to facilitate glucose uptake by the cells. As long as insulin over-production by the pancreas is sufficient to overcome the insulin resistance, blood glucose levels typically stay within a normal healthy range. However, over time, the pancreas can no longer produce enough insulin and glucose levels in the blood rise above the normal range, leading to the development of T2D.

[0004] T2D is thought to result from a combination of genetic and environmental factors. The risk of developing T2D is greatly increased when associated with factors, such as high blood pressure, overweight or obesity, insufficient physical activity, poor diet, and an “apple shaped” body where extra weight is carried around the waist. T2D can often initially be managed with healthy eating and regular physical activity. However, over time most people with T2D will also need medications and insulin.

[0005] T2D is associated with a number of co-morbidities and complications, including hypertension, dyslipidemia, cardiovascular diseases, blindness and eye problems, and increased risk of heart attacks, strokes, damage to kidneys, and limb amputations.

[0006] Glucose transport in skeletal muscle is mediated by the glucose transport protein 4 (Glut4). Glut4s are normally stored on intracellular vesicles, which fuse with the plasma membrane in response to insulin stimulation or muscle contraction. Skeletal muscle contains a specialized transverse-tubule (TT) network that facilitates the transmission of electrical signalfrom the neurons to control calcium release and muscle contraction. The TT network is also the active site for fusion of Glut4-containing vesicles with the plasma membrane for glucose uptake into the skeletal muscle. Studies have shown that exercise enhances Glut4-translocation to TT and sarcolemma membrane, thereby improving glucose uptake into muscle fibers. The challenge is that patients with diabetes often encounter difficulty with physical activity and exercise due to fragility. Physical inactivity further increases the risk of obesity and metabolic disease. There has been a growing interest in identifying “molecular transducers” that induce the metabolic benefits of exercise via pharmacologic or biologic means. SUMMARY OF THE DISCLOSURE

[0007] Provided are nucleic acids comprising micro RNA (miRNA) binding sequences from a 3′ untranslated region (3′ UTR) of a Mitsugumin 29 (MG29) mRNA, which miRNA binding sequences when delivered to a cell can bind miRNAs that otherwise would bind to and inhibit MG29 mRNA translation and production of MG29 protein. MG29 protein plays essential roles in control of calcium signaling and glucose uptake into skeletal muscle and maintenance of and / or increase in MG29 protein expression increases glucose uptake into skeletal muscle and decreases blood glucose levels. The miRNA binding nucleotide sequences from the 3’ UTR of a MG29 mRNA include, e.g., miR-29a-3p binding nucleotide sequences, miR-181a-5p binding nucleotide sequences, miR-375-3p binding nucleotide sequences, and miR-29a2-3p binding nucleotide sequences. In certain embodiments, the miRNA sequences are connected by linkers. In some embodiments, a nucleic acid comprises multiple copies, e.g., 3 copies of one miRNA binding nucleotide sequence connected by linkers. In some embodiments, a nucleic acid comprises one copy of one miRNA binding nucleotide sequence and one copy of a second miRNA binding nucleotide sequence connected by a linker. In some embodiments, a nucleic acid comprises multiple copies of one miRNA binding nucleotide sequence and one copy of a second miRNA binding nucleotide sequence connected by linkers.

[0008] In some embodiments, a nucleic acid comprises multiple copies, e.g., three copies of one miRNA binding nucleotide sequence and multiple copies, e.g., three copies of a second miRNA binding nucleotide sequence connected by linkers. In some embodiments, the linkers comprise four nucleotides, e.g., aatt. In some embodiments, the linkers can comprise up to 20 nucleotides. In some embodiments, the multiple copies of a miRNA binding nucleotide sequence are arranged adjacent to each other in a nucleic acid. In some embodiments, the multiple copies of each miRNA binding nucleotide sequence are present in the nucleic acid inan interspersed arrangement, e.g., copies of one miRNA binding nucleotide sequence are not arranged strictly adjacent.

[0009] Also provided are nucleic acids comprising a miRNA binding sequence from a 3’ UTR of a MG29 mRNA and an inverted terminal repeat, e.g., an adeno-associated virus inverted terminal repeat (AAV ITR). In some embodiments, provided is a nucleic acid comprising a single miRNA binding nucleotide sequence and an AAV ITR. In some embodiments, provided is a nucleic acid comprising multiple copies of a single miRNA binding nucleotide sequence and an AAV ITR. In some embodiments, provided is a nucleic acid comprising different miRNA binding nucleotide sequences and an AAV ITR. In some embodiments, provided is a nucleic acid comprising multiple copies of different miRNA binding nucleotide sequence and an AAV ITR.

[0010] Further provided are vectors and viral particles, e.g., AAV particles comprising a nucleic acid described or a vector described herein encapsulated in a viral capsid, e.g., an AAV capsid. In some aspects, provided are compositions and kits comprising a nucleic acid described herein, a vector described herein, or a viral particle, e.g., an AAV particle described for administration to a subject in need thereof for the treatment of a metabolic disease, e.g., diabetes and / or obesity. Also provided are methods of increasing glucose uptake in muscle, e.g., skeletal muscle, by administering a nucleic acid, vector, viral particle, e.g., AAV particle or a composition described herein to a subject in need thereof. The methods provided increase glucose uptake into a muscle cell, e.g., a skeletal muscle cell and reduce blood glucose and treat a metabolic disorder, e.g., diabetes and / or obesity.

[0011] In some embodiments, the nucleic acid comprises a miRNA binding nucleotide sequence that comprises a miR binding site from a 3′ UTR of an MG29 mRNA. In certain embodiments, the miRNA binding nucleotide sequences provided herein comprise between about 20 and about 25 nucleotides, e.g., 23 nucleotides. In some embodiments, a nucleic acid provided herein comprises more than one miRNA binding nucleotide sequence from a 3′ UTR of an MG29 mRNA. In some embodiments, a nucleic acid comprises multiple copies of a miRNA binding nucleotide sequence from a 3′ UTR of an MG29 mRNA. In some embodiments, a nucleic acid comprises multiple copies of more than one miRNA binding nucleotide sequence from a 3′ UTR of an MG29 mRNA.

[0012] In another aspect, provided is a nucleic acid comprising at least two different miRNA binding nucleotide sequences of a 3′ UTR of a MG29 mRNA.

[0013] In another aspect, provided is a nucleic acid comprising at least one miRNA binding nucleotide sequence of a 3′ UTR of a MG29 mRNA and an inverted terminal repeat.

[0014] In another aspect, provided is a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-59 or 62-65.

[0015] In another aspect, provided is a vector comprising a nucleic acid described herein.

[0016] In another aspect, provided is a composition comprising a nucleic acid described herein or a vector described herein.

[0017] In some aspect, provided is a viral particle comprising a nucleic acid described herein, or a vector described herein.

[0018] In some embodiments, the viral particle is an adeno-associated virus (AAV) particle.

[0019] In another aspect, provided are methods of increasing MG29 expression in a cell, the method comprising contacting the cell with an effective amount of: a nucleic acid described herein, a vector described herein, a composition described herein, or a viral particle, e.g., an AAV particle described herein.

[0020] In another aspect, a provided is a method of increasing glucose uptake in a cell of a subject in need thereof, the method comprising contacting the cell of the subject with an effective amount of: a nucleic acid described herein, a vector described herein, a composition described herein, or a viral particle, e.g., an AAV particle described herein.

[0021] In another aspect, provided is a method of increasing insulin sensitivity in a subject in need thereof, the method comprising administering to the subject an effective amount of: a nucleic acid described herein, a vector described herein, a composition described herein, or a viral particle, e.g., an AAV particle described herein.

[0022] In another aspect, provided is a method of reducing blood glucose in a subject in need thereof, the method comprising administering to the subject an effective amount of: a nucleic acid described herein, a vector described herein, a composition described herein, or a viral particle, e.g., an AAV particle described herein.

[0023] In another aspect, provided is a method of treating a metabolic disease (e.g., diabetes) in a subject in need thereof, the method comprising administering to the subject with an effective amount of: a nucleic acid described herein, a vector described herein, or a composition described herein, or a viral particle, e.g., an AAV particle described herein.

[0024] In another aspect, provided is a method of increasing MG29 expression in skeletal muscle of a subject in need thereof comprising contacting at least one nucleic acid, that is at least 16 nucleotides in length and has at least 90% sequence identity to a 16 nucleotide sequence of the MG293′UTR, with the MG293′ UTR of the subject.

[0025] In another aspect, provided is a method of increasing MG29 expression in skeletal muscle of a subject in need thereof comprising contacting a skeletal muscle cell with a nucleicacid comprising at least two miRNA binding nucleotide sequences each comprising at least 23 nucleotides and having at least 90% identity to a nucleotide sequence of SEQ ID NO: 1-7.

[0026] In some embodiments, the nucleic acid is a complementary DNA (cDNA) having at least 90% homology to a sequence of a miRNA molecule described herein.

[0027] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65 or a miRNA binding nucleotide sequence having at least 90% identity to a sequence of a miRNA binding nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65.

[0028] In another aspect, provided is a method of improving glucose uptake, regulating glycolysis, or treating a metabolic disorder, e.g., type 2 diabetes and / or obesity in a patient comprising administering to the patient at least one nucleic acid, that is at least 16 nucleotides in length and has at least 90% sequence identity to a 16 nucleotide sequence of the M29 3′UTR, with the MG293′ UTR of the patient.

[0029] In another aspect, provided is a method of improving glucose uptake, regulating glycolysis, or treating a metabolic disease, e.g., type 2 diabetes and / or obesity in a patient comprising administering to the patient at least two miRNA binding nucleotide sequences of any one of SEQ ID NO: 1-59 or 62-65 or a miRNA binding nucleotide sequence having at least 90% identity to a sequence of a miRNA binding nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65.

[0030] In another aspect, provided is a nucleic acid described herein, a vector described herein, a composition described herein, or a viral particle, e.g., an AAV particle, described herein, for use in the treatment of a metabolic disease, e.g., type 2 diabetes and / or obesity.

[0031] In another aspect, provided is a kit comprising a nucleic acid described herein, a vector described herein, a composition described herein, or a viral particle, e.g., an AAV particle described herein.

[0032] The details of one or more embodiments of the present disclosure are set forth herein. Other features, objects, and advantages of the present disclosure will be apparent from the Detailed Description, Examples, Figures, and Claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0034] FIG.1. Schematics of the 3′ UTR of human SYPL2 (e.g. MG29) and mouse MG29, which contains binding sites for miRNAs.

[0035] FIG.2A. Mg29- / -mice develop glucose intolerance compared to wild type mice. FIG.2B. Mg29- / -mice develop insulin resistance compared to wild type mice. Squares – wild type; gray circles – MG29- / -. Similar results were previous disclosed in the granted patent (US Patent 8,603,993, issued December 10, 2013, refer to Figure 5C and 6). FIG.2C. AAV- mediated MG29 overexpression improves glucose handling in wild type mice. Dark gray circles – MG29 AAV.

[0036] FIG.3. Exercise induces MG29 expression in mouse skeletal muscle. Compared with the basal level of MG29, after two hour of exercise training of wild type mice, MG29 protein was measurable increased, whereas the expression level of MG53 (a muscle specific- protein) remained unchanged following exercise.

[0037] FIGs.4A to 4B. The coding sequence (CDS) of MG29 lacking a 3′ UTR is expressed in C2C12 cells. FIG.4A. Human and mouse MG29 protein expression is severely suppressed in the presence of a 3′ UTR in the plasmid. FIG.4B. Transient overexpression of the 3′ UTR in C2C12 cells uncovers endogenous MG29 protein expression in a dose- dependent manner.

[0038] FIGs.5A to 5C. Expression of miRNAs and MG29 and MG53 in differentiated C2C12 cells. FIG.5A. Levels of MG29 mRNA increase in differentiated C2C12 cells (day 0, 3, 5, and 7 of differentiation). Similarly, levels of MG53 mRNA (skeletal muscle specific gene) increase during differentiation. FIG.5B. Western Blot showing undetectable MG29 protein, whereas MG53 increases during differentiation of C2C12 cells. FIG.5C. The expression of miR-29a-3p, -375-3p, and -181a-5p is unchanged in undifferentiated (Day 0) and differentiated (Day 5) C2C12 cells by stem-loop RT-qPCR (n = 3, data are presented as mean ± SD). Sno202 was utilized as an internal miRNA control. The values given are the normalized values of the mean.

[0039] FIGs.6A to 6B. Constructs with 3′ UTR miRNA binding sequences inserted down- stream of a Renilla luciferase reporter. FIG.6A. Schematic of cloned construct. FIG.6B. Relative Renilla luciferase / firefly luciferase activity in C2C12 cells 48 hours post-transfection showing proof of concept that engineered miR-binding sequences present in a 3′ UTR of Renilla luciferase reduce Renilla luciferase expression. Lower levels of measured Renilla / luciferase activity indicated enhanced binding of miRs to the miR-sponge and BS-miR- 29. Relative expression levels were normalized to the pSiCHECK2 and mutant controls. (n = 3,data are presented as mean ± SD). Note: *BS indicates binding site on MG293′ UTR, *mut indicates a mutant scrambled negative control.

[0040] FIGs.7A to7B. Schematic of two different miR-Sponge constructs. FIG.7A. The CMV promoter drives RFP expression as a visualization control. The U6 plasmid drives ubiquitous expression of the miR-sponge. The construct includes Puromycin resistance as a selective pressure marker. FIG.7B. Refined construct with a skeletal muscle specific CK8e (partial promoter sequences of creatine kinase), tMCK (triple tandem of muscle creatine kinase), or MHCK7 (a chimeric promoter of muscle creatine kinase and alpha-myosin heavy chain) promoter driving eGFP or Glut4 expression in skeletal muscle. The CMV promoter is utilized for puromycin resistance.

[0041] FIGs.8A to 8B. Expression of constructs in C2C12 myotubes. FIG.8A. RFP expression under CMV promoter in Day 4 differentiated C2C12 myotubes after electroporation with the pU6-miR sponge plasmid. RFP expression indicates transfection efficiency as well as reports on specific cells which express the delivered miR-Sponge. FIG.8B. eGFP expression under the tMCK promoter after electroporation. Day 5 differentiated C2C12 myotubes show enhanced expression compared to Day 0. eGFP serves as a reporter for electroporation efficiency and of cells that express the miR-Sponge.

[0042] FIGs.9A to 9C. Schematic design of the AAV9-miRSponge expression cassettes for complementary gene transfer, promoting glucose uptake, or reducing plasma membrane damage using the MHCK7 promoter. FIG.9A. Construct utilizing the MHCK7 skeletal muscle specific promoter, eGFP serves as a reporter for delivery. FIG.9B. Construct utilizing the MHCK7 promoter, Glut4 serves as a complementary component delivered to aid in the amount of glucose uptake into skeletal muscle following miR-sponge mediated overexpression of MG29. FIG.9C. Construct utilizing the MHCK7 promoter, driving tPA-MG53. tPA-MG53 is designed to achieve sustained elevation of circulating MG53 by fusing a tPA secretory peptide at the N-terminus of the MG53 protein. This serves as a complementary gene delivery system to aid in enhanced damage repair experienced by those with diabetes.

[0043] FIG.10. Enhanced MG29 expression in C2C12 myotubes after transfection with miR-Sponges. Western Blot of Day 5 differentiated C2C12 cells after transfection of single or combination miR-Sponge constructs utilizing pU6. Notably, MG29 protein expression was enhanced in miR-sponge combinations, while only slightly increased with miR-Sponge targeting the individual regions.

[0044] FIGs.11A to 11B. Intramuscular injection of MyoAAV-hMG29 in HDF mice. FIG.12A. Western Blot of human and mouse MG29 expression. FIG.11B. [18F] FDG PETimaging in HFD mice showing muscle glucose dynamics in hMG29-injected and non-injected skeletal muscle.

[0045] FIGs.12A to 12B. Intramuscular injection of MyoAAV-hMG29 and MyoAAV- Sponge in HDF mice. FIG.12A. [18F] FDG PET imaging in HDF mice showing muscle glucose dynamics in MyoAAV-hMG29 injected and MyoAAV-Sponge injected skeletal muscle. FIG.12B. [18F] FDG uptake expressed as SUVbw in contralateral non-injected muscle (squares, control) or MyoAAV-Sponge injected skeletal muscle (circles).

[0046] FIG.13 Intramuscular injection of MyoAAV-EGFP (EGFP), MyoAAV-hMG29 (hMG29), and MyoAAV-Sponge (Sponge) in HDF mice; percent body weight (top graph) and percent fasting glucose (bottom graph).

[0047] FIG.14. Western Blot of MG29 expression in C2C12 myotubes infected with MyoAAV-EGFP, a triple miRNA binding nucleotide sequence MyoAAV-Sponge, and a quadruple miRNA binding nucleotide sequence MyoAAV-Sponge. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE DISCLOSURE

[0048] Provided are nucleic acids comprising miRNA binding nucleotide sequences that are useful for increasing the expression of MG29 and treating a metabolic disease, e.g., diabetes and / or obesity. In some aspects, provided are nucleic acids comprising a miRNA binding nucleotide sequence that binds to miRNAs, e.g., miRNAs that interact with the 3′ UTR of an MG29 mRNA. In some embodiments, the nucleic acids comprise more than one miRNA binding nucleotide sequence. In some embodiments, the nucleic acids comprise miRNA binding nucleotide sequences that bind the same miRNA. In some embodiments, the nucleic acids comprise miRNA binding nucleotide sequences that bind different miRNAs, e.g., different miRNAs that interact with the 3′ UTR of a MG29 mRNA. In some embodiments, the nucleic acids are present in a recombinant adeno associated virus (AAV). Nucleic Acids Comprising miRNA Binding Nucleotide Sequences

[0049] Provided are nucleic acids comprising miRNA binding nucleotide sequences that bind miRNAs and are useful for increasing the expression of a MG29 protein in a cell. Without wanting to be bound by a particular theory, it is hypothesized that the miRNA binding nucleotide sequences reduce the level of miRNAs present in a cell and, thereby, prevent and / or reduce the miRNA-mediated inhibition of translation of MG29 protein.

[0050] The terms “miRNA” and “miR” are used interchangeably herein and refer to micro RNAs, which are short nucleotides that bind to mRNA molecules, preferably in a 3′ UTR region and inhibit translation of the mRNA.

[0051] The term “miRNA binding nucleotide sequence,” as used herein, refers to a sequence of nucleotides that is sufficiently complementary with a sequence of nucleotides in a miRNA to effect base pairing between the miRNA and the miRNA binding nucleotide sequence. Typically, the miRNA binding nucleotide sequence and the miRNA are sufficiently complementary to base pair, thereby inhibiting binding of the miRNA to a target mRNA, e.g., the 3′ UTR of the MG29 mRNA. The terms “complementary” or “complementarity,” as used herein, refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick or non-traditional base pairing. In reference to the nucleic acid molecules described herein the binding free energy for nucleic acid molecules with their miRNA target or complementary sequence is sufficient to allow, e.g., inhibition of miRNA binding to a target mRNA. A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds with another nucleic acid (e.g., 5, 6, 7, 8, 9, or 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). The term “perfectly complementary,” means that all the contiguous residues of a nucleic acid sequence hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In some embodiments, nucleic acids provided herein have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity. In some embodiments, a miRNA binding nucleotide sequence comprises between about 15 and about 25 nucleotides that are complementary to a miRNA. In some embodiments, a miRNA binding nucleotide sequence comprises between about 7 and about 25 nucleotides that are complementary to a miRNA and additional nucleotides that are non-complementary to the miRNA. In some embodiments, a miRNA binding nucleotide sequence described herein comprises about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides that are complementary to a miRNA that binds in a 3’ UTR of a MG29 mRNA.

[0052] In some aspects, provided are miRNA sponges. The term “miRNA sponge,” as used herein refers to a nucleic acid comprising at least two miRNA binding nucleotide sequences. In some embodiments, a miRNA sponge comprises between about 2 and about 100 miRNA binding nucleotide sequences. A miRNA sponge described herein when present in a cell comprising a miRNA that binds the miRNA binding nucleotide sequence binds such miRNAand prevents the miRNA from binding to its natural target, e.g., a miRNA binding sequence in a 3’ UTR of a mRNA, e.g., a MG29 mRNA.

[0053] In some embodiments, a nucleic acid comprises about 1 to about 100 miRNA binding nucleotide sequences. In some embodiments, a nucleic acid comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 30, between 30 and 40, between 40 and 50, between 50 and 70, or between 70 and 100 miRNA binding nucleotide sequences. In some embodiments, the about 2 to about 100 miRNA binding nucleotide sequences are linked by linkers. In some embodiments, a nucleic acid comprises at least two different miRNA binding nucleotide sequences. In some embodiments, the length of a miRNA binding nucleotide sequence may depend on the length and / or sequence of the miRNA to which it binds.

[0054] In some embodiments, a miRNA binding nucleotide sequence comprises about 4 nucleotides to about 100 nucleotides, about 5 nucleotides to about 90 nucleotides, about 6 nucleotides to about 80 nucleotides, about 7 nucleotides to about 70 nucleotides, about 8 nucleotides to about 60 nucleotides, about 9 nucleotides to about 50 nucleotides, about 10 nucleotides to about 40 nucleotides, or about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 25 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, or about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, about 50 nucleotides, about 51 nucleotides, about 52 nucleotides, about 53 nucleotides, about 54 nucleotides, about 55 nucleotides, about 56 nucleotides, about 57 nucleotides, about 58 nucleotides, about 59 nucleotides, about 60 nucleotides, about 61 nucleotides, about 62 nucleotides, about 63 nucleotides, about 64 nucleotides, about 65 nucleotides, about 66nucleotides, about 67 nucleotides, about 68 nucleotides, about 69 nucleotides, about 70 nucleotides, about 71 nucleotides, about 72 nucleotides, about 73 nucleotides, about 74 nucleotides, about 75 nucleotides, about 76 nucleotides, about 77 nucleotides, about 78 nucleotides, about 79 nucleotides, about 80 nucleotides, about 81 nucleotides, about 82 nucleotides, about 83 nucleotides, about 84 nucleotides, about 85 nucleotides, about 86 nucleotides, about 87 nucleotides, about 88 nucleotides, about 89 nucleotides, about 90 nucleotides, about 91 nucleotides, about 92 nucleotides, about 93 nucleotides, about 94 nucleotides, about 95 nucleotides, about 96 nucleotides, about 97 nucleotides, about 98 nucleotides, about 99 nucleotides, or about 100 nucleotides, or more.

[0055] The nucleic acids described herein do not comprise a natural MG29 mRNA and / or a 3’ UTR of a natural MG29 mRNA. A nucleic acid described herein is not a natural MG29 mRNA sequence and / or a 3’ UTR sequence of a natural MG29 mRNA.

[0056] In some embodiments, a nucleic acid sequence comprises a miRNA binding portion and a portion that does not bind a miRNA, e.g., a sequence that comprises a linker. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence and, optionally, comprises one or more non-miRNA-binding sequences e.g., a sequence that comprises a linker. In some embodiments, a nucleic acid sequence comprises a miRNA binding nucleotide sequence and an adjacent non-miRNA-binding nucleotide sequence, e.g., a linker sequence. In some embodiments, a nucleic acid sequence comprises a miRNA binding nucleotide sequence and at least two non-miRNA-binding nucleotide sequences, e.g., linker sequences.

[0057] In some aspects, provided are nucleic acids comprising a plurality of binding nucleotide sequences that bind different miRNAs. In some embodiments, a nucleic acid comprises a plurality of binding nucleotide sequences that bind different miRNAs, wherein at least one of the miRNA binding nucleotide sequences is present in multiple copies. In some embodiments, a nucleic acid comprises a plurality of binding nucleotide sequences that bind different miRNAs, wherein all of the miRNA binding nucleotide sequences are present in multiple copies.

[0058] In some embodiments, a nucleic acid comprises a plurality of binding nucleotide sequences that bind different miRNAs, wherein the plurality of miRNA binding nucleotide sequences is present in a specific order in the nucleic acid. In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of at least one miRNA binding nucleotide sequence, which multiple copies are present adjacent to each other in the nucleic acid. In some embodiments, a nucleic acid comprising a plurality ofmiRNA binding nucleotide sequences comprises multiple copies of at least two of the miRNA binding nucleotide sequences, which multiple copies are present adjacent to each other in the nucleic acid. In some embodiments, a nucleic acid comprising a plurality of binding nucleotide sequences comprises multiple copies of at least three of the miRNA binding nucleotide sequences, which multiple copies are present adjacent to each other in the nucleic acid. In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of each of the plurality of binding nucleotide sequences present adjacent to each other in the nucleic acid.

[0059] In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of each of the miRNA binding nucleotide sequences, wherein multiple copies of one miRNA binding nucleotide sequence are interspersed with multiple copies of another miRNA binding nucleotide sequence. For example, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of miRNA binding nucleotide sequences 1, 2, 3, and 4, wherein in the nucleic acid from 5′ to 3′, e.g., a miRNA binding nucleotide sequence 1 is followed by a miRNA binding nucleotide sequence 2, which is followed by a miRNA binding nucleotide sequence 3, which is followed by a miRNA binding nucleotide sequence 4. In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of miRNA binding nucleotide sequences 1, 2, 3, and 4, wherein in the nucleic acid from 5′ to 3′, e.g., a miRNA binding nucleotide sequence 1 is followed by a miRNA binding nucleotide sequences 3, which is followed by a miRNA binding nucleotide sequence 4, which is followed by a miRNA binding nucleotide sequence 2. In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of miRNA binding nucleotide sequences 1, 2, 3, and 4 in the order of 1, 2, 4, 3; or 1, 3, 2, 4; or 1, 4, 2, 3; or 1, 4, 3, 2. In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of miRNA binding nucleotide sequences 1, 2, 3, and 4 in the order of 2, 1, 3, 4; 2, 1, 4, 3; 2, 3, 1, 4; 2, 3, 4, 1; 2, 4, 1, 3; or 2, 4, 3, 1. In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of miRNA binding nucleotide sequences 1, 2, 3, and 4 in the order of 3, 1, 2, 4; 3, 1, 4, 2; 3, 2, 1, 4; 3, 2, 4, 1; 3, 4, 1, 2; or 3, 4, 2, 1. In some embodiments, a nucleic acid comprising a plurality of miRNA binding nucleotide sequences comprises multiple copies of miRNA binding nucleotide sequences 1, 2, 3, and 4 in the order of 4, 1, 2, 3; 4, 1, 3, 2; 4, 2, 1, 3; 4, 2, 3, 1; 4, 3, 1, 2; or 4, 3, 2, 1.

[0060] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleotide sequence that binds a miRNA-29a from the 3′ arm of a pre-miRNA-29a (miRNA-29a-3p). In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleotide sequence that binds a miRNA-29a from the 5′ arm of a pre-miRNA- 29a (miRNA-29a-5p). In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of miRNA-29a-3p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a-3p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a miRNA-29a-3p. In some embodiments, a nucleic acid sequence comprises more than one copy of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a-3p.

[0061] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has about 1 to about 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 1. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has 1, 2, 3, 4 or 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 1.

[0062] In some embodiments, a nucleic acid sequence comprises between about two and about 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a-3p.

[0063] In some embodiments, a nucleic acid sequence comprises between 2 and 5 copies, between 2 and 10 copies, between two and 20 copies, between three and 45 copies, between four and 40 copies, between 5 and 35 copies, between 6 and 30 copies, between 7 and 25 copies, between 8 and 20 copies, between 9 and 15 copies; or about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, abut 14, about 15, about 16, about 17, about 18, about 19, between 20 and 22, between 22 and 24, between 24 and 26, between 26 and 28, between 28 and 30, between 30 and 32, between 32 and 34, between 34 and 36, between 36 and 38, between 38 and 40, between 40 and 42, between 42 and 44, between 44and 46, between 46 and 48, between 48 and 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a- 3p.

[0064] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleic sequence binding a miRNA-181a-5p from a 5′ arm of a pre-miRNA-181a (miRNA-181a-5p). In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleic sequence binding a miRNA-181a-3p from a 3′ arm of a pre-miRNA-181a (miRNA-181a-3p). In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of miRNA-181a-5p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-181a-5p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a miRNA-181a-5p. In some embodiments, a nucleic acid sequence comprises more than one copy of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-181a- 5p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has about 1 to about 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 2. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has 1, 2, 3, 4 or 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 2In some embodiments, a nucleic acid sequence comprises between about two and about 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-181a-5p. In some embodiments, a nucleic acid sequence comprises between 2 and 5 copies, between 2 and 10 copies, between 2 and 20 copies, between three and 45 copies, between four and about 40 copies, between 5 and 35 copies, between 6 and 30 copies, between 7 and 25 copies, between 8 and 20 copies, between 9 and 15 copies; or about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, abut 14, about 15, about 16, about 17, about 18, about 19, between 20 and 22, between 22 and 24, between 24 and 26,between 26 and 28, between 28 and 30, between 30 and 32, between 32 and 34, between 34 and 36, between 36 and 38, between 38 and 40, between 40 and 42, between 42 and 44, between 44 and 46, between 46 and 48, between 48 and 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-181a-5p.

[0065] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleic sequence binding a miRNA-375-3p from a 3′ arm of a pre-miRNA-375 (miRNA- 375-3p). In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleic sequence binding a miRNA-375-5p from a 5′ arm of a pre-miRNA-375 (miRNA-375- 5p). In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of miRNA-375-3p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-375-3p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a miRNA-375-3p. In some embodiments, a nucleic acid sequence comprises more than one copy of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-375-3p.

[0066] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has about 1 to about 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 3. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has 1, 2, 3, 4 or 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 3

[0067] In some embodiments, a nucleic acid sequence comprises between about two and about 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-375-3p.

[0068] In some embodiments, a nucleic acid sequence comprises between 2 and 5 copies, between 2 and 10 copies, between 2 and 20 copies, between three and 45 copies, between four and 40 copies, between 5 and about 35 copies, between 6 and 30 copies, between 7 and 25copies, between 8 and 20 copies, between 9 and 15 copies; or about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, abut 14, about 15, about 16, about 17, about 18, about 19, between 20 and 22, between 22 and 24, between 24 and 26, between 26 and 28, between 28 and 30, between 30 and 32, between 32 and 34, between 34 and 36, between 36 and 38, between 38 and 40, between 40 and 42, between 42 and 44, between 44 and 46, between 46 and 48, between 48 and 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-375- 3p.

[0069] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleic sequence binding a miRNA-29a2 from a 3′ arm of a pre-miRNA-29a (miRNA- 29a2-3p). In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 80% identical to a nucleic sequence binding a miRNA-29a2 from a 5′ arm of a pre-miRNA-29a (miRNA- 29a2-5p). In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a nucleotide sequence of miRNA-29a2-3p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a2-3p. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a miRNA-29a2-3p. In some embodiments, a nucleic acid sequence comprises more than one copy of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a2-3p.

[0070] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has about 1 to about 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 4. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that has 1, 2, 3, 4 or 5 nucleotide changes, e.g., nucleotide substitutions compared to a sequence of a miRNA binding nucleotide sequences of SEQ ID NO: 4

[0071] In some embodiments, a nucleic acid sequence comprises between about two and about 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a2-3p. In some embodiments, a nucleic acidsequence comprises between 2 and 5 copies, between 2 and 10 copies, between 2 and 20 copies, between three and 45 copies, between four and 40 copies, between 5 and 35 copies, between 6 and 30 copies, between 7 and 25 copies, between 8 and 20 copies, between 9 and 15 copies; or about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, abut 14, about 15, about 16, about 17, about 18, about 19, between 20 and 22, between 22 and 24, between 24 and 26, between 26 and 28, between 28 and 30, between 30 and 32, between 32 and 34, between 34 and 36, between 36 and 38, between 38 and 40, between 40 and 42, between 42 and 44, between 44 and 46, between 46 and 48, between 48 and 50 copies of a miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a2-3p.

[0072] In some embodiments, a nucleic acid sequence comprises a leader sequence. The term “leader sequence,” as used herein, refers to a nucleotide sequence that is located at the 5′ end of a nucleic acid described herein and is important for transport of the nucleic acid into a cell nucleus and / or for regulation of translation of the nucleic acid.

[0073] In some embodiments, at least one of the at least two miRNA binding nucleotide sequences does not extend more than 10 bases beyond the 3’ or 5’ end of SEQ ID NO: 2.

[0074] In some embodiments, at least one of the at least two miRNA binding nucleotide sequences does not extend more than 10 bases beyond the 3’ or 5’ end of SEQ ID NO: 3.

[0075] In some embodiments, at least one of the at least two miRNA binding nucleotide sequences does not extend more than 10 bases beyond the 3’ or 5’ end of SEQ ID NO: 5.

[0076] In some embodiments, at least one of the at least two miRNA binding nucleotide sequences does not extend more than 10 bases beyond the 3’ or 5’ end of SEQ ID NO: 6.

[0077] In some embodiments, at least one of the at least two miRNA binding nucleotide sequences does not extend more than 10 bases beyond the 3’ or 5’ end of SEQ ID NO: 7.

[0078] In some embodiments, the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 2 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO: 2.

[0079] In some embodiments, the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 3 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO: 3.

[0080] In some embodiments, the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 5 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO: 5.

[0081] In some embodiments, the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 6 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO: 6.

[0082] In some embodiments, the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 7 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO: 7. Nucleic Acids with miRNA Binding Sequences and Viral Inverted Terminal Repeats

[0083] In some embodiments, provided are nucleic acids comprising a single miRNA binding nucleotide sequence. In some embodiments, a nucleic acid comprises a single miRNA binding nucleotide sequence that is present in the nucleic acid in multiple copies. In some embodiments, a nucleic acid comprises a single miRNA binding nucleotide sequence and an inverted terminal repeat. In some embodiments, the inverted terminal repeat is an adeno- associated virus inverted terminal repeat (AAV ITR). In some embodiments, a nucleic acid comprises a single miRNA binding nucleotide sequence and two AAV ITRs. In some embodiments, a nucleic acid comprises a single miRNA binding nucleotide sequence flanked by two AAV ITRs.

[0084] In some embodiments, a nucleic acid comprises multiple copies of a single miRNA binding nucleotide sequence and an AAV ITR. In some embodiments, a nucleic acid comprises multiple copies of a single miRNA binding nucleotide sequence and two AAV ITRs. In some embodiments, a nucleic acid comprises multiple copies of a single miRNA binding nucleotide sequence flanked by two AAV ITRs.

[0085] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a-3p and an AAV ITR. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a-3p and two AAV ITRs. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a- 3p flanked by two AV ITRs.

[0086] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-181a-5p and an AAV ITR. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotidesequence that is at least 90% identical to a miRNA-181a-5p and two AAV ITRs. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-181a- 5p flanked by two AV ITRs.

[0087] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-375-3p and an AAV ITR. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-375-3p and two AAV ITRs. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-375- 3p flanked by two AV ITRs.

[0088] In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a2-3p and an AAV ITR. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a2-3p and two AAV ITRs. In some embodiments, a nucleic acid sequence comprises at least one miRNA binding nucleotide sequence that comprises a nucleotide sequence that is at least 90% identical to a miRNA-29a2- 3p flanked by two AV ITRs.

[0089] In some embodiments, the at least two different miRNA binding nucleotide sequences are selected from the group consisting of a miRNA-29a from the 3′ arm of a precursor (miR-29a-3p) binding nucleotide sequence or a sequence having at least 90% identity to a miR-29a-3p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA; a miRNA-181a from the 5′ arm of a precursor (miR-181a-5p) binding nucleotide sequence or a sequence having at least 90% identity to a miR-181a-5p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA; a miRNA-375 from the 3′ arm of a precursor (miR-375-3p) binding nucleotide sequence of the 3′ UTR of the MG29 mRNA or a sequence having at least 90% identity to a miR-375-3p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA; and a miRNA-29a2 from the 3′ arm of a precursor (miR-29a2-3p) binding nucleotide sequence of the 3′ UTR of the MG29 mRNA or a sequence having at least 90% identity to a miR-29a2-3p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA.

[0090] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotidesequence of SEQ ID NO: 1. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a complement of a nucleotide sequence of SEQ ID NO: 1. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 1. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 1. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 1. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 1, which nucleotide sequence is part of a vector, e.g., a plasmid or a viral vector. In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 1.

[0091] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 1.In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 2. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a complement of a nucleotide sequence of SEQ ID NO:2. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 2. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 2. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 2. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 2, which nucleotide sequence is part of a vector, e.g., a plasmid or a viral vector. In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 2.

[0092] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 2.

[0093] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 3. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a complement of a nucleotide sequence of SEQ ID NO: 3. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 3. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 3. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 3.In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 3.

[0094] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 3.

[0095] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 4. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a complement of a nucleotide sequence of SEQ ID NO: 4. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 4. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 4. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 4.

[0096] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences, wherein at least one of the miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 4. In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 4.

[0097] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a complement of a nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65. In some embodiments, a nucleic acid comprises a nucleotide sequence of any one of SEQ ID NO: 1-59 or 62-65. In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 5.

[0098] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences wherein at least one of the miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 5.

[0099] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences, wherein at least one of the miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 6.

[0100] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences, wherein at least one of the miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 6.

[0101] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences, wherein at least one of the miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 7.

[0102] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences, wherein at least one of the miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 7.

[0103] In some embodiments, the nucleic acid further comprises a leader sequence.

[0104] In some embodiments, the nucleic acid further comprises a linker sequence. In some embodiments, the linker sequence comprises at least 1 nucleotide. In some embodiments, the linker sequence comprises between about 1 nucleotide and about 20 (e.g., between 1 and 2, between 3 and 5, between 6 and 10, between 11 and 15, or between 16 and 20) nucleotides. In some embodiments, a nucleic acid comprises a linker sequence between every miRNA binding nucleotide sequence. For example, in some embodiments, a nucleic acid comprising three miRNA binding nucleotide sequences comprises a linker sequence between the first and the second miRNA binding nucleotide sequence and between the second and the third miRNA binding nucleotide sequence.

[0105] In some embodiments, the nucleic acid comprises between about 3 copies and about 100 copies of at least one miRNA binding nucleotide sequence.

[0106] In some embodiments, a nucleic acid comprises at least two different miRNA binding nucleotide sequences that comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2.

[0107] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 25 or 26.

[0108] In some embodiments, the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3.

[0109] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 17 or 18.

[0110] In some embodiments, the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4.

[0111] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 21 or 22.

[0112] In some embodiments, the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2 and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3.

[0113] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 23 or 24.

[0114] In some embodiments, the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2 and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4.

[0115] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 63 or 64.

[0116] In some embodiments, the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3 and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4.

[0117] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 19 or 20.

[0118] In some embodiments, the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2 and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4.

[0119] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 63 or 64.

[0120] In some embodiments, the nucleic acid comprises at least three different miRNA binding nucleotide sequences.

[0121] In some embodiments, the at least three different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3.

[0122] In some embodiments, the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 27 or 28.

[0123] In some embodiments, the at least three different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4.

[0124] In some embodiments, the at least three miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 29 or 30.

[0125] In some embodiments, the at least three different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, anucleotide sequence that is at least 90% identical to SEQ ID NO: 3, and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4.

[0126] In some embodiments, the at least three miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 65 or 66.

[0127] In some embodiments, the nucleic acid comprises four different miRNA binding nucleotide sequences.

[0128] In some embodiments, the four different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4.

[0129] In some embodiments, the four miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 31 or 32.

[0130] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 5-8 and 10-18. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 5-8 and 10-18. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 5-8 and 10-18. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 5-8 and 10-18.

[0131] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 9 and 18-24. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 9 and 18-24. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 9 and 18-24. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 9 and 18-24.

[0132] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 25-30 and 41-44. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 25-30 and 41-44. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 25-30 and 41-44. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 25-30 and 41-44.

[0133] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 31-40. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 31-40. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 31-40. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 31-40.

[0134] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 45-48. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 45-48. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 45-48. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 45-48.

[0135] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 49-52. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 49-52. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 49-52. In some embodiments, a nucleic acid a nucleotide sequence of SEQ ID NO: 49-52.

[0136] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 53 and 54. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 53 and 54. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 53 and 54. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 53 and 54.

[0137] In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to a nucleotide sequence of SEQ ID NO: 55 and 56. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO: 55 and 56. In some embodiments, a nucleic acid comprises a miRNA binding nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 55 and 56. In some embodiments, a nucleic acid comprises a nucleotide sequence of SEQ ID NO: 55 and 56. Linkers

[0138] In some embodiments, a nucleic acid comprises at least two miRNA binding nucleotide sequences connected by a linker.

[0139] The term “linker,” as used herein, refers to a moiety that fuses together two or more molecules into a single molecule. In some embodiments, a linker connects two or more nucleotide sequences. In some embodiments, a linker is a non-nucleotide linker. In some embodiments, a linker is optionally substituted C1-100(e.g., C1-10, C10-20, C20-40, or C40-100)alkylene, optionally substituted C2-100(e.g., C2-10, C10-20, C20-40, or C40-100) alkenylene, optionally substituted C2-100 (e.g., C2-10, C10-20, C20-40, or C40-100) alkynylene, optionally substituted C1-100(e.g., C1-10, C10-20, C20-40, or C40-100) heteroalkylene, optionally substituted C2-100 (e.g., C2-10, C10-20, C20-40, or C40-100) heteroalkenylene, or optionally substituted C2-100 (e.g., C2-10, C10-20, C20-40, or C40-100) heteroalkynylene; and optionally wherein one or more (e.g., 2, 3, or 4) backbone atoms of the C1-100 (e.g., C1-10, C10-20, C20-40, or C40-100) alkylene, C2-100 (e.g., C2- 10, C10-20, C20-40, or C40-100) alkenylene, C2-100 (e.g., C2-10, C10-20, C20-40, or C40-100) alkynylene, C1-100 (e.g., C1-10, C10-20, C20-40, or C40-100) heteroalkylene, C2-100 (e.g., C2-10, C10-20, C20-40, or C40-100) heteroalkenylene, or C2-100 (e.g., C2-10, C10-20, C20-40, or C40-100) heteroalkynylene are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene, as valency permits. In some embodiments, a linker comprises an ethylene glycol oligomer (e.g., between a dimer and a tetramer, between a tetramer and an octamer, between an octamer and a dodecamer, or between a dodecamer and a 20-mer). In some embodiments, a linker comprises nucleotides A, T, G, C, and combinations thereof. In some embodiments, a linker comprises nucleotides A and T.

[0140] The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 100 carbon atoms (“C1–100alkyl”). In some embodiments, an alkyl group has 1 to 20 carbon atoms (“C1–20 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1–12 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1–10alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1–9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1–8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1–7alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1–6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1–5alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1–4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1–3alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1–2alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6alkyl”). Examples of C1–6alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert- butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2- butanyl, tert-amyl), and hexyl (C6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), n-dodecyl (C12), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) orsubstituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as fluorine). In certain embodiments, the alkyl group is an unsubstituted C1–12 alkyl (such as unsubstituted C1–6alkyl, e.g., −CH3(Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec- butyl (sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is a substituted C1–12 alkyl (such as substituted C1–6 alkyl, e.g., –CH2F, –CHF2, –CF3, –CH2CH2F, –CH2CHF2, –CH2CF3, or benzyl (Bn)).

[0141] The term “heteroalkyl” refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, sulfur, and phosphorous within (e.g., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 100 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–100alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–20alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–12alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 11 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–11 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–10alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–9alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–8alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–7alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–6 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC1–5 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC1–4alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC1–3alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC1–2alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC1alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC2-6 alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents (e.g., oxo, substituted or unsubstituted C1-6 alkyl (e.g., –CH3)). In certain embodiments, the heteroalkyl group is an unsubstituted heteroC1–12 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC1–12 alkyl. In some embodiments, unsubstituted heteroC1alkyl is –OCH3or –CH2OH. In some embodiments, substituted heteroC1 alkyl is –C(=O)NH2. In some embodiments, unsubstituted heteroC2 alkyl is – OCH2CH3, –CH2OCH3, or –CH2CH2OH. The terms “heteroCz1-z2alkyl” and “Cz1-z2heteroalkyl” are used interchangeably, wherein each of z1 and z2 is independently an integer.

[0142] The term “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 100 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 1 to 100 carbon atoms (“C1-100 alkenyl”). In some embodiments, an alkenyl group has at least 2 carbon atoms. In some embodiments, an alkenyl group has 1 to 20 carbon atoms (“C1-20alkenyl”). In some embodiments, an alkenyl group has 1 to 12 carbon atoms (“C1–12 alkenyl”). In some embodiments, an alkenyl group has 1 to 11 carbon atoms (“C1–11 alkenyl”). In some embodiments, an alkenyl group has 1 to 10 carbon atoms (“C1–10alkenyl”). In some embodiments, an alkenyl group has 1 to 9 carbon atoms (“C1–9 alkenyl”). In some embodiments, an alkenyl group has 1 to 8 carbon atoms (“C1–8alkenyl”). In some embodiments, an alkenyl group has 1 to 7 carbon atoms (“C1–7 alkenyl”). In some embodiments, an alkenyl group has 1 to 6 carbon atoms (“C1–6alkenyl”). In some embodiments, an alkenyl group has 1 to 5 carbon atoms (“C1–5 alkenyl”). In some embodiments, an alkenyl group has 1 to 4 carbon atoms (“C1–4alkenyl”). In some embodiments, an alkenyl group has 1 to 3 carbon atoms (“C1–3 alkenyl”). In some embodiments, an alkenyl group has 1 to 2 carbon atoms (“C1–2alkenyl”). In some embodiments, an alkenyl group has 1 carbon atom (“C1 alkenyl”). In certain embodiments, an alkenyl group is C2–3 alkenyl, C2–4 alkenyl, C2–5 alkenyl, C2–6 alkenyl, C2–7 alkenyl, C2–8 alkenyl, C2–9alkenyl, C2–10C2–12alkenyl, C2–16alkenyl, C2–20alkenyl, C2–30alkenyl, C2–40 alkenyl, C2–50 alkenyl, C2–60 alkenyl, C2–70 alkenyl, C2–80 alkenyl, C2–90 alkenyl, or C2–100 alkenyl. The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) orterminal (such as in 1-butenyl). Examples of C1–4alkenyl groups include methylidenyl (C1), ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C1–6alkenyl groups include the aforementioned C2-4alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C1-20 alkenyl. In certain embodiments, the alkenyl group is a substituted C1-20 alkenyl. In an alkenyl group, a C=C double bond for which the stereochemistry is not specified (e.g., −CH=CHCH3 or ) may be in the (E)- or (Z)- configuration.

[0143] The term “heteroalkenyl” refers to an alkenyl group, at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, sulfur, and phosphorous within (e.g., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 100 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–100 alkenyl”). In some embodiments, a heteroalkenyl group has at least 2 carbon atoms. In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–20 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 12 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–12alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 11 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–11alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–10 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–9alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–8alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–7 alkenyl”). In some embodiments, a heteroalkenyl group has 1to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1–6 alkenyl”). In some embodiments, aheteroalkenyl group has 1 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1–5 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1–4 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC1–3alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 2 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC1–2 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1–6 alkenyl”). In certain embodiments, a heteroalkenyl group is C2–3heteroalkenyl, C2–4heteroalkenyl, C2–5heteroalkenyl, C2–6heteroalkenyl, C2–7 heteroalkenyl, C2–8 heteroalkenyl, C2–9 heteroalkenyl, C2–10 heteroalkenyl, C2–12heteroalkenyl, C2–16heteroalkenyl, C2–20heteroalkenyl, C2–30heteroalkenyl, C2–40heteroalkenyl, C2–50 heteroalkenyl, C2–60 heteroalkenyl, C2–70 heteroalkenyl, C2–80 heteroalkenyl, C2–90heteroalkenyl, or C2–100heteroalkenyl. Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents (e.g., oxo, substituted or unsubstituted C1-6 alkyl (e.g., –CH3)). In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC1–20alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC1–20 alkenyl. In some embodiments, unsubstituted heteroC1 alkenyl is –CH=NH or =N–CH3. The terms “heteroCz1-z2 alkenyl” and “Cz1-z2 heteroalkenyl” are used interchangeably, wherein each of z1 and z2 is independently an integer.

[0144] The term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 100 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C1-100alkynyl”). In some embodiments, an alkynyl group has 1 to 20 carbon atoms (“C1-20 alkynyl”). In some embodiments, an alkynyl group has at least 2 carbon atoms. In some embodiments, an alkynyl group has 1 to 10 carbon atoms (“C1-10alkynyl”). In some embodiments, an alkynyl group has 1 to 9 carbon atoms (“C1-9 alkynyl”). In some embodiments, an alkynyl group has 1 to 8 carbon atoms (“C1-8alkynyl”). In some embodiments, an alkynyl group has 1 to 7 carbon atoms (“C1-7 alkynyl”). In some embodiments, an alkynyl group has 1 to 6 carbon atoms (“C1-6 alkynyl”). In some embodiments, an alkynyl group has 1 to 5 carbon atoms (“C1-5alkynyl”). In some embodiments, an alkynyl group has 1 to 4 carbon atoms (“C1-4 alkynyl”). In some embodiments, an alkynyl group has 1 to 3 carbon atoms (“C1-3alkynyl”). In someembodiments, an alkynyl group has 1 to 2 carbon atoms (“C1-2alkynyl”). In some embodiments, an alkynyl group has 1 carbon atom (“C1 alkynyl”). In certain embodiments, an alkynyl group is C2–3alkynyl, C2–4alkynyl, C2–5alkynyl, C2–6alkynyl, C2–7alkynyl, C2–8alkynyl, C2–9 alkynyl, C2–10 alkynyl, C2–12 alkynyl, C2–16 alkynyl, C2–20 alkynyl, C2–30 alkynyl, C2–40alkynyl, C2–50alkynyl, C2–60alkynyl, C2–70alkynyl, C2–80alkynyl, C2–90alkynyl, or C2–100alkynyl. The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C1-4 alkynyl groups include, without limitation, methylidynyl (C1), ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C1-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C1-20alkynyl. In certain embodiments, the alkynyl group is a substituted C1-20 alkynyl.

[0145] The term “heteroalkynyl” refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, sulfur, and phosphorous within (e.g., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 100 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1–100alkynyl”). In some embodiments, a heteroalkynyl group has at least 2 carbon atoms. In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1–20 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1–10 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1–9 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1–8 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1–7alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1–6alkynyl”). In some embodiments, aheteroalkynyl group has 1 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1–5 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 4 carbon atoms, at least one triple bond, and 1or 2 heteroatoms within the parent chain (“heteroC1–4 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC1–3alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 2 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC1–2 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1–6 alkynyl”). In certain embodiments, a heteroalkynyl group is C2–3heteroalkynyl, C2–4heteroalkynyl, C2–5heteroalkynyl, C2–6heteroalkynyl, C2–7heteroalkynyl, C2–8 heteroalkynyl, C2–9 heteroalkynyl, C2–10 heteroalkynyl, C2–12 heteroalkynyl, C2–16heteroalkynyl, C2–20heteroalkynyl, C2–30heteroalkynyl, C2–40heteroalkynyl, C2–50heteroalkynyl, C2–60 heteroalkynyl, C2–70 heteroalkynyl, C2–80 heteroalkynyl, C2–90 heteroalkynyl, or C2–100heteroalkynyl. Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents (e.g., oxo, substituted or unsubstituted C1-6 alkyl (e.g., –CH3)). In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC1–20alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC1–20 alkynyl. In some embodiments, unsubstituted heteroC1 alkynyl is – C≡N. The terms “heteroCz1-z2 alkynyl” and “Cz1-z2 heteroalkynyl” are used interchangeably, wherein each of z1 and z2 is independently an integer.

[0146] The term “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 13 ring carbon atoms (“C3-13 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 12 ring carbon atoms (“C3-12carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 11 ring carbon atoms (“C3-11 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C3-10carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C3-7carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-6carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5to 10 ring carbon atoms (“C5-10carbocyclyl”). Exemplary C3-6carbocyclyl groups include cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-8 carbocyclyl groups include the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3-10 carbocyclyl groups include the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. Exemplary C3-8carbocyclyl groups include the aforementioned C3-10carbocyclyl groups as well as cycloundecyl (C11), spiro[5.5]undecanyl (C11), cyclododecyl (C12), cyclododecenyl (C12), cyclotridecane (C13), cyclotetradecane (C14), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C3-14carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-14 carbocyclyl.

[0147] In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C3-10cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10cycloalkyl”). Examples of C5-6cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4).Examples of C3-8cycloalkyl groups include the aforementioned C3-6cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C3-14cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C3-14 cycloalkyl. In certain embodiments, the carbocyclyl includes 0, 1, or 2 C=C double bonds in the carbocyclic ring system, as valency permits.

[0148] The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14- membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3–14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3–14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3–14 membered heterocyclyl. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valency permits.

[0149] In some embodiments, a heterocyclyl group is a 5–10 membered non-aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5–8 membered non-aromatic ring system havingring carbon atoms and 1–4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5–6 membered non-aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–6 membered heterocyclyl”). In some embodiments, the 5–6 membered heterocyclyl has 1–3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5–6 membered heterocyclyl has 1–2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5–6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0150] Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include aziridinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H- furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3- b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4- tetrahydro-1,6-naphthyridinyl, and the like.

[0151] The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6–14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10aryl”; e.g., naphthyl such as 1–naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6-14 aryl. In certain embodiments, the aryl group is a substituted C6-14aryl.

[0152] The term “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continues to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In certain embodiments, the heteroaryl is substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. Incertain embodiments, the heteroaryl is substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.

[0153] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1–3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1–2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

[0154] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0155] The term “halo” or “halogen” refers to fluorine (fluoro, −F), chlorine (chloro, −Cl), bromine (bromo, −Br), or iodine (iodo, −I).

[0156] The term “alkoxy” refers to an -O-alkyl substituent.

[0157] Affixing the suffix “-ene” to a group indicates the resulting group is a polyvalent (e.g., divalent, trivalent, or tetravalent) moiety. For example, alkylene is a polyvalent moiety of alkyl, alkenylene is a polyvalent moiety of alkenyl, alkynylene is a polyvalent moiety of alkynyl, heteroalkylene is a polyvalent moiety of heteroalkyl, heteroalkenylene is a polyvalent moiety of heteroalkenyl, heteroalkynylene is a polyvalent moiety of heteroalkynyl, carbocyclylene is a polyvalent moiety of carbocyclyl, heterocyclylene is a polyvalent moiety of heterocyclyl, arylene is a polyvalent moiety of aryl, and heteroarylene is a polyvalent moiety of heteroaryl. In some embodiments, unsubstituted C1 heteroalkylene is –OCH2– or –CH2O–. In some embodiments, substituted C1heteroalkylene is –NHC(=O)– or –C(=O)NH–. In some embodiments, unsubstituted C2 heteroalkylene is –OCH2CH2– or –CH2CH2O–. In some embodiments, unsubstituted C4heteroalkylene is –(OCH2CH2)2– or –(CH2CH2O)2–. In some embodiments, unsubstituted C6 heteroalkylene is –(OCH2CH2)3– or –(CH2CH2O)3–. In some embodiments, unsubstituted C8 heteroalkylene is –(OCH2CH2)4– or –(CH2CH2O)4–. In some embodiments, unsubstituted C10heteroalkylene is –(OCH2CH2)5– or –(CH2CH2O)5–. In some embodiments, unsubstituted C12 heteroalkylene is –(OCH2CH2)6– or –(CH2CH2O)6–.

[0158] A group is optionally substituted unless expressly provided otherwise. The term “optionally substituted” refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted. “Optionally substituted” refers to a group which is substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does notspontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds and includes any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety. The disclosure is not limited in any manner by the exemplary substituents described herein.

[0159] Exemplary carbon atom substituents include halogen, −CN, −NO2, −N3, −SO2H, −SO3H, −OH, −ORaa, −ON(Rbb)2, −N(Rbb)2, −N(Rbb)3+X−, −N(ORcc)Rbb, −SH, −SRaa, −SSRcc, 2, 32, 4,−B(ORcc)2, −BRaa(ORcc), C1–20alkyl, C1–20perhaloalkyl, C1–20alkenyl, C1–20alkynyl, heteroC1–20 alkyl, heteroC1–20 alkenyl, heteroC1–20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups; wherein X−is a counterion; or two geminal hydrogens on a carbon atom are replaced with the group =O, =S, =NN(Rbb)2, =NNRbbC(=O)Raa, =NNRbbC(=O)ORaa, =NNRbbS(=O)2Raa, =NRbb, or =NORcc; each instance of Raais, independently, selected from C1–20alkyl, C1–20perhaloalkyl, C1–20 alkenyl, C1–20 alkynyl, heteroC1–20 alkyl, heteroC1–20alkenyl, heteroC1–20alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14aryl, and 5-14 membered heteroaryl, or twoRaagroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups; each instance of Rbbis, independently, selected from hydrogen, −OH, −ORaa, −N(Rcc)2, −CN, −C(=O)Raa, −C(=O)N(Rcc)2, −CO2Raa, −SO2Raa, −C(=NRcc)ORaa, −C(=NRcc)N(Rcc)2, −SO2N(Rcc)2, −SO2Rcc, −SO2ORcc, −SORaa, −C(=S)N(Rcc)2, −C(=O)SRcc, −C(=S)SRcc, −P(=O)(Raa)2,−P(=O)(ORcc)2,−P(=O)(N(Rcc)2)2, C1–20alkyl, C1–20perhaloalkyl, C1–20alkenyl, C1–20 alkynyl, heteroC1–20alkyl, heteroC1–20alkenyl, heteroC1–20alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14aryl, and 5-14 membered heteroaryl, or two Rbbgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups; each instance of Rccis, independently, selected from hydrogen, C1–20alkyl, C1–20perhaloalkyl, C1–20 alkenyl, C1–20 alkynyl, heteroC1–20 alkyl, heteroC1–20 alkenyl, heteroC1–20 alkynyl, C3-10carbocyclyl, 3-14 membered heterocyclyl, C6-14aryl, and 5-14 membered heteroaryl, or two Rccgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups; each instance of Rddis, independently, selected from halogen, −CN, −NO2, −N3, −SO2H, −SO3H, −OH, −ORee, −ON(Rff)2, −N(Rff)2, −N(Rff)3+X−, −N(ORee)Rff, −SH, −SRee, −SSRee, −C(=O)Ree, −CO2H, −CO2Ree, −OC(=O)Ree, −OCO2Ree, −C(=O)N(Rff)2, −OC(=O)N(Rff)2, −NRffC(=O)Ree, −NRffCO2Ree, −NRffC(=O)N(Rff)2, −C(=NRff)ORee, −OC(=NRff)Ree, −OC(=NRff)ORee, −C(=NRff)N(Rff)2, −OC(=NRff)N(Rff)2, −NRffC(=NRff)N(Rff)2, −NRffSO2Ree, −SO2N(Rff)2, −SO2Ree, −SO2ORee, −OSO2Ree, −S(=O)Ree, −Si(Ree)3, −OSi(Ree)3, −C(=S)N(Rff)2, −C(=O)SRee, −C(=S)SRee, −SC(=S)SRee, −P(=O)(ORee)2, −P(=O)(Ree)2, −OP(=O)(Ree)2, −OP(=O)(ORee)2, C1–10 alkyl, C1–10 perhaloalkyl, C1–10alkenyl, C1–10alkynyl, heteroC1–10alkyl, heteroC1–10alkenyl, heteroC1–10alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, and 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgggroups, or two geminal Rddsubstituents are joined to form =O or =S; wherein X−is a counterion;each instance of Reeis, independently, selected from C1–10alkyl, C1–10perhaloalkyl, C1–10 alkenyl, C1–10 alkynyl, heteroC1–10 alkyl, heteroC1–10 alkenyl, heteroC1–10 alkynyl, C3-10 carbocyclyl, C6-10aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgggroups; each instance of Rffis, independently, selected from hydrogen, C1–10 alkyl, C1–10 perhaloalkyl, C1–10 alkenyl, C1–10 alkynyl, heteroC1–10 alkyl, heteroC1–10 alkenyl, heteroC1–10 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, and 5-10 membered heteroaryl, or two Rffgroups are joined to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgggroups; each instance of Rggis, independently, halogen, −CN, −NO2, −N3, −SO2H, −SO3H, −OH, −OC1–6 alkyl, −ON(C1–6 alkyl)2, −N(C1–6 alkyl)2, −N(C1–6 alkyl)3+X−, −NH(C1–6 alkyl)2+X−, −NH2(C1–6alkyl)+X−, −NH3+X−, −N(OC1–6alkyl)(C1–6alkyl), −N(OH)(C1–6alkyl), −NH(OH), −SH, −SC1–6 alkyl, −SS(C1–6 alkyl), −C(=O)(C1–6 alkyl), −CO2H, −CO2(C1–6 alkyl), −OC(=O)(C1–6alkyl), −OCO2(C1–6alkyl), −C(=O)NH2, −C(=O)N(C1–6alkyl)2, −OC(=O)NH(C1–6 alkyl),−NHC(=O)( C1–6 alkyl),−N(C1–6 alkyl)C(=O)( C1–6 alkyl), −NHCO2(C1–6 alkyl), −NHC(=O)N(C1–6 alkyl)2, −NHC(=O)NH(C1–6 alkyl), −NHC(=O)NH2, −C(=NH)O(C1–6alkyl), −OC(=NH)(C1–6alkyl), −OC(=NH)OC1–6alkyl, −C(=NH)N(C1–6alkyl)2, −C(=NH)NH(C1–6 alkyl), −C(=NH)NH2, −OC(=NH)N(C1–6 alkyl)2, −OC(NH)NH(C1–6 alkyl), −OC(NH)NH2, −NHC(NH)N(C1–6alkyl)2, −NHC(=NH)NH2, −NHSO2(C1–6alkyl), −SO2N(C1–6 alkyl)2, −SO2NH(C1–6 alkyl), −SO2NH2, −SO2C1–6 alkyl, −SO2OC1–6 alkyl, −OSO2C1–6alkyl, −SOC1–6alkyl, −Si(C1–6alkyl)3, −OSi(C1–6alkyl)3−C(=S)N(C1–6alkyl)2, C(=S)NH(C1–6 alkyl), C(=S)NH2, −C(=O)S(C1–6 alkyl), −C(=S)SC1–6 alkyl, −SC(=S)SC1–6 alkyl, −P(=O)(OC1–6alkyl)2, −P(=O)(C1–6alkyl)2, −OP(=O)(C1–6alkyl)2, −OP(=O)(OC1–6alkyl)2, C1–10 alkyl, C1–10 perhaloalkyl, C1–10 alkenyl, C1–10 alkynyl, heteroC1–10 alkyl, heteroC1–10alkenyl, heteroC1–10alkynyl, C3-10carbocyclyl, C6-10aryl, 3-10 membered heterocyclyl, or 5- 10 membered heteroaryl; or two geminal Rggsubstituents can be joined to form =O or =S; and each X−is a counterion.

[0160] In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl, −ORaa, −SRaa, −N(Rbb)2, –CN, –SCN, –NO2, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, −OC(=O)Raa,−OCO2Raa, −OC(=O)N(Rbb)2, −NRbbC(=O)Raa, −NRbbCO2Raa, or −NRbbC(=O)N(Rbb)2. In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1–10alkyl, −ORaa, −SRaa, −N(Rbb)2, – CN, –SCN, –NO2, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, −OC(=O)Raa, −OCO2Raa, −OC(=O)N(Rbb)2, −NRbbC(=O)Raa, −NRbbCO2Raa, or −NRbbC(=O)N(Rbb)2, wherein Raais hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1–10 alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl) when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each Rbbis independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1–10 alkyl, or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6alkyl, −ORaa, −SRaa, −N(Rbb)2, –CN, –SCN, or –NO2. In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen moieties) or unsubstituted C1–10 alkyl, −ORaa, −SRaa, −N(Rbb)2, –CN, –SCN, or –NO2, wherein Raais hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1–10alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl) when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each Rbbis independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1–10alkyl, or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts).

[0161] In certain embodiments, each nitrogen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6alkyl, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, or a nitrogen protecting group. In certain embodiments, each nitrogen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10alkyl,−C(=O)Raa,−CO2Raa,−C(=O)N(Rbb)2, or a nitrogen protecting group, wherein Raais hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10alkyl, or an oxygen protecting group when attached to an oxygen atom; and each Rbbis independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10alkyl, or a nitrogen protecting group. In certainembodiments, each nitrogen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl or a nitrogen protecting group.

[0162] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to herein as an “amino protecting group”). Nitrogen protecting groups include −OH, −ORaa, −N(Rcc)2, −C(=O)Raa, −C(=O)N(Rcc)2, −CO2Raa, −SO2Raa, −C(=NRcc)Raa, −C(=NRcc)ORaa, −C(=NRcc)N(Rcc)2, −SO2N(Rcc)2, −SO2Rcc, −SO2ORcc, −SORaa, −C(=S)N(Rcc)2, −C(=O)SRcc, −C(=S)SRcc, C1–10 alkyl (e.g., aralkyl, heteroaralkyl), C1–20 alkenyl, C1–20 alkynyl, hetero C1–20 alkyl, hetero C1–20 alkenyl, hetero C1–20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups, and wherein Raa, Rbb, Rcc, and Rddare as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999.

[0163] For example, in certain embodiments, at least one nitrogen protecting group is an amide group (e.g., a moiety that include the nitrogen atom to which the nitrogen protecting groups (e.g., −C(=O)Raa) is directly attached). In certain such embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3- phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N’-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o- phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o- nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o- (benzoyloxymethyl)benzamide.

[0164] In certain embodiments, at least one nitrogen protecting group is a carbamate group (e.g., a moiety that include the nitrogen atom to which the nitrogen protecting groups (e.g., −C(=O)ORaa) is directly attached). In certain such embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of methyl carbamate, ethyl carbamate, 9- fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7- dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2- phenylethyl carbamate (hZ), 1–(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl- 2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl- 2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1- (3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1- isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3- dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4- dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m- chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5- benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4- dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p- decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N- dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p’-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1- methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5- dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl- 1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p- (phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4- (trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0165] In certain embodiments, at least one nitrogen protecting group is a sulfonamide group (e.g., a moiety that include the nitrogen atom to which the nitrogen protecting groups (e.g., −S(=O)2Raa) is directly attached). In certain such embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6- trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′- dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0166] In certain embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N’-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N- acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N- dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4- tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5- triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2- (trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro- 2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4- methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4- methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N’- oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2- pyridyl)mesityl]methyleneamine, N-(N’,N’-dimethylaminomethylene)amine, N-p- nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2- hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1- cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N- [phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys). In some embodiments, two instances of a nitrogen protecting group together with the nitrogen atoms to which the nitrogen protecting groups are attached are N,N’-isopropylidenediamine. In certain embodiments, at least one nitrogen protecting group is Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts.

[0167] In certain embodiments, each oxygen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10alkyl, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, or an oxygen protecting group. In certain embodiments, each oxygen atom substituents is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, or an oxygen protecting group, wherein Raais hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or an oxygen protecting group when attached to an oxygen atom; and each Rbbis independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or a nitrogen protecting group. In certain embodiments, each oxygen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6alkyl or an oxygen protecting group.

[0168] In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an “hydroxyl protecting group”). Oxygen protecting groups include −Raa, −N(Rbb)2, −C(=O)SRaa, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, −C(=NRbb)Raa, −C(=NRbb)ORaa, −C(=NRbb)N(Rbb)2, −S(=O)Raa, −SO2Raa, −Si(Raa)3, −P(Rcc)2, −P(Rcc)3+X−, −P(ORcc)2, −P(ORcc)3+X−, −P(=O)(Raa)2, −P(=O)(ORcc)2, and −P(=O)(N(Rbb) 2)2, wherein X−, Raa, Rbb, and Rccare as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999.

[0169] In certain embodiments, each oxygen protecting group, together with the oxygen atom to which the oxygen protecting group is attached, is selected from the group consisting of methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl(GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4- methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2- yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1- benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2- trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4- picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p’-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, 4,4′-dimethoxytrityl (4,4′-dimethoxytriphenylmethyl or DMT), α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4’- bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 4,4’- Dimethoxy-3"‘-[N-(imidazolylmethyl) ]trityl Ether (IDTr-OR), 4,4’-Dimethoxy-3"‘-[N- (imidazolylethyl)carbamoyl]trityl Ether (IETr-OR), 1,1-bis(4-methoxyphenyl)-1′- pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3- benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t- butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4- methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p- nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl carbonate (MTMEC-OR), 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6- dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N’,N’-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate , benzylsulfonate, and tosylate (Ts).

[0170] In certain embodiments, at least one oxygen protecting group is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl.

[0171] In certain embodiments, each sulfur atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, or a sulfur protecting group. In certain embodiments, each sulfur atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10alkyl, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, or a sulfur protecting group, wherein Raais hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or an oxygen protecting group when attached to an oxygen atom; and each Rbbis independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or a nitrogen protecting group. In certain embodiments, each sulfur atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl or a sulfur protecting group.

[0172] In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a “thiol protecting group”). In some embodiments, each sulfur protecting group is selected from the group consisting of −Raa, −N(Rbb)2, −C(=O)SRaa, −C(=O)Raa, −CO2Raa, −C(=O)N(Rbb)2, −C(=NRbb)Raa, −C(=NRbb)ORaa, −C(=NRbb)N(Rbb)2, −S(=O)Raa, −SO2Raa, −Si(Raa)3, −P(Rcc)2, −P(Rcc)3+X−, −P(ORcc)2, −P(ORcc)3+X−, are as definedin detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999.

[0173] In certain embodiments, the molecular weight of a substituent is lower than 250, lower than 200, lower than 150, lower than 100, or lower than 50 g / mol. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, nitrogen, and / or silicon atoms. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, and / or nitrogen atoms. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, and / or iodine atoms. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, and / or chlorine atoms. In certain embodiments, a substituent comprises 0, 1, 2, or 3 hydrogen bond donors. In certain embodiments, a substituent comprises 0, 1, 2, or 3 hydrogen bond acceptors.

[0174] In some embodiments, a linker comprises nucleotides AATT. In some embodiments, a linker consists of nucleotides AATT.

[0175] In some embodiments, a linker comprises at least one nucleotide. In some embodiments, a linker comprises between about one nucleotide and about 20 nucleotides. In some embodiments, a linker comprises about 1 nucleotide to about 8 nucleotides, or about 2 nucleotides to about 7 nucleotides, or about 3 nucleotides to about 6 nucleotides, about 1 nucleotide to about 3 nucleotides, about 3 nucleotides to about 6 nucleotides, about 6 nucleotides to about 9 nucleotides, about 9 nucleotides to about 14 nucleotides, or about 14 nucleotides to about 20 nucleotides; or about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, or about 20 nucleotides, or more.

[0176] In some embodiments, a nucleic acid comprises multiple copies of a miRNA binding nucleotide sequence, wherein each of the multiple copies of miRNA binding nucleotide sequences is connected to an adjacent miRNA binding nucleotide sequence by a linker comprising between about 1 and about 20 nucleotides.

[0177] In some embodiments, a nucleic acid comprises a plurality of different miRNA binding nucleotide sequences, wherein each of the plurality of different miRNA binding nucleotide sequences is connected to an adjacent miRNA binding nucleotide sequence by a linker comprising between about 1 and about 20 nucleotides.

[0178] In some embodiments, a nucleic acid comprises at least two different miRNA binding nucleotide sequences connected by a linker. In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by a samelinker. In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers. In some embodiments, the different linkers have different lengths. For examples, in some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers where one linker comprises about 1 nucleotide while a second linker comprises about 20 nucleotides. In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers where one linker comprises about 1 nucleotide, a second linker comprises about 20 nucleotides, and a third linker comprises about 10 nucleotides. In some embodiments, a nucleic acid can comprise any combination of linkers, each linker comprising between about 1 nucleotide and about 20 nucleotides.

[0179] In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers, wherein a linker of a specific length, e.g., 8 nucleotides connects multiple copies of a same miRNA binding nucleotide sequence. In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers where a linker of a specific length connects two specific miRNA binding nucleotide sequences. In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers where a linker of a specific length connects multiple copies of two specific miRNA binding nucleotide sequences.

[0180] In some embodiments, the different linkers have different nucleotide compositions. For examples, in some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers where one linker comprises A and T nucleotides and a second linker comprises A and G nucleotides. In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences connected by different linkers where one linker comprises A and T nucleotides, a second linker comprises A and G nucleotides, and a third linker comprises T and C nucleotides. In some embodiments, a nucleic acid can comprise any combination of linkers comprising any combination of A, T, G, and C nucleotides. Regulatory Elements

[0181] In some embodiments, a nucleic acid comprises more than two different miRNA binding nucleotide sequences and a regulatory element. A regulatory element refers to a nucleotide sequence or structural component of a nucleic acid which is involved in theregulation of expression of components of the nucleic acid (e.g., a miRNA binding nucleotide sequence). Regulatory elements include, but are not limited to, promoters, enhancers, silencers, insulators, response elements, initiation sites, termination signals, and ribosome binding sites.

[0182] Promoters include constitutive promoters, inducible promoters, tissue-specific promoters, cell type-specific promoters, and synthetic promoters. For example, a nucleic acid described herein may include viral promoters or promoters from mammalian genes that are generally active in promoting transcription. Non-limiting examples of constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A and cytomegalovirus (CMV) promoters. Non-limiting examples of constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the β-actin promoter.

[0183] Inducible promoters or other inducible regulatory elements may also be used to achieve desired levels of a mRNA binding nucleotide sequence in a cell. Non-limiting examples of suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, and metallothionein genes. Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline.

[0184] In some embodiments, a tissue-specific promoter or other tissue-specific regulatory element is used in a nucleic acid comprising a miRNA binding nucleotides sequence.

[0185] In some embodiments, a nucleic acid described herein comprises a promoter for expression in a muscle tissue. In some embodiments, a nucleic acid described herein comprises a promoter for expression in a skeletal muscle. In some embodiments, a nucleic acid described herein comprises a skeletal muscle specific CK8e (partial promoter sequences of creatine kinase) promoter, a tMCK (triple tandem of muscle creatine kinase) promoter, or a MHCK7 (a chimeric promoter of muscle creatine kinase and alpha-myosin heavy chain) promoter.

[0186] Synthetic promoters are also contemplated herein. A synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like. Recombinant Vectors

[0187] In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence is present in a nucleic acid vector. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence is present in a plasmid. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence ispresent in a recombinant viral genome, e.g., a recombinant AAV genome. In some embodiments, a nucleic acid comprises at least one miRNA binding nucleotide sequence and an AAV ITR. In some embodiments, a nucleic acid comprises at least one miRNA binding nucleotide sequence and a 5′ AAV ITR. In some embodiments, an ITR is a 5′ ITR, i.e., an ITR that is 5′ from a nucleic acid sequence that comprises at least one miRNA binding nucleotide sequence in a nucleic acid vector (e.g., a recombinant AAV genome).

[0188] In some embodiments, a nucleic acid comprises at least one miRNA binding nucleotide sequence and a 3′ AAV ITR. In some embodiments, an ITR is a 3′ ITR, i.e., an ITR that is 3′ from a nucleic acid sequence that comprises at least one miRNA binding nucleotide sequence in a nucleic acid vector (e.g., a recombinant AAV genome).

[0189] In some embodiments, a nucleic acid comprises at least one miRNA binding nucleotide sequence and a 5′ AAV ITR and a 3′ AAV ITR.

[0190] An ITR serves as an origin of replication and is comprised of two arm palindromes (B-B' and C-C') embedded in a larger stem palindrome (A-A', stem A-domain), and a single stranded sequence (D-sequence) (see e.g., Mroske, et al., Human Gene Therapy Methods 23(2):128-36 (2012); Yan, et al., “Inverted Terminal Repeat Sequences Are Important for Intermolecular Recombination and Circularization of Adeno-Associated Virus Genomes” J Virol.79(1):364-379 (2005)).

[0191] In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence comprises a 5′ ITR and a 3′ ITR. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence is present within a recombinant viral genome (e.g., a recombinant AAV genome)comprising a 5′ ITR and a 3′ ITR.

[0192] In some embodiments, a vector further comprises regulatory elements as described herein.

[0193] In some embodiments, a recombinant viral genome (e.g., a recombinant AAV genome) is encapsidated within a viral capsid, e.g., an AAV capsid forming a viral particle, e.g., an AAV particle. In some embodiments, a nucleic acid described herein is encapsidated within a wild-type AAV capsid or another AAV capsid described herein, such as an AAV capsid comprising one or more amino acid substitutions and / or a derivative or hybrid of a wild- type AAV capsid.

[0194] In some embodiments, a nucleic acid (e.g., a recombinant AAV genome) comprises native AAV genes or native AAV nucleotide sequences. In some embodiments, one or more native AAV genes or native AAV nucleotide sequences may be removed from a nucleic acid (e.g., a recombinant AAV genome). In some embodiments, one or more native AAV genes ornative AAV nucleotide sequences may be removed from a nucleic acid (e.g., a recombinant AAV genome) and replaced with a nucleic acid comprising at least one miRNA binding nucleotide sequence. In some embodiments, a nucleic acid (e.g., a recombinant AAV genome) comprises native AAV genes or native AAV nucleotide sequences and a nucleic acid comprising at least one miRNA binding nucleotide sequence.

[0195] In some embodiments, a nucleic acid vector (e.g., a recombinant AAV genome) can be of any AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or a combination of serotypes. In some embodiments, a nucleic acid (e.g., a recombinant AAV genome) encapsidated within an AAV capsid forms a pseudotyped AAV particle, such that the genome is of a serotype distinct from the capsid in which it is encapsidated. For example, a nucleic acid vector (e.g., a recombinant AAV genome) of serotype AAV2 may be encapsidated within a capsid of serotype AAVrh74.

[0196] As described herein, the 5′ ITR refers to the ITR at the 5' terminus of the nucleic acid (e.g., recombinant AAV genome), and the 3′ ITR refers to the ITR at the 3' terminus of the nucleic acid (e.g., recombinant AAV genome). Each ITR in its native or wild-type form is or is about 145 nucleotides in length (e.g., about 140 nucleotides, about 145 nucleotides, about 150 nucleotides, about 155 nucleotides, about 160 nucleotides, or about 165 nucleotides) and comprises a D-sequence and a stem A-domain. Each ITR can independently be of any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74), or both ITRs may be of the same serotype. ITRs are described, for example, in Grimm et al. J. Virol.80(1):426-439 (2006). An example of a 5′ ITR of AAV2 is provided below. A 3′ ITR has a nucleotide sequence which is the reverse complement of the corresponding 5′ ITR (e.g., the AAV23′ ITR has a nucleotide sequence which is the reverse complement of the AAV25′ ITR).

[0197] Example of wild-type AAV25′ ITR: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGA CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC TCCATCACTAGGGGTTCCT (SEQ ID NO: 66)

[0198] In some embodiments, modifications of a nucleic acid (e.g., a recombinant AAV genome) result in the generation of a recombinant viral genome (e.g., a recombinant AAV genome)that is self-complementary (scAAV) and possesses a single AAV ITR. While a single- stranded recombinant AAV genomes has a packaging capacity of about 4.7 kb, a self- complementary AAV genome has a limited packaging capacities of about 2.3 kb.

[0199] In some embodiments, a nucleic acid (e.g., a recombinant AAV genome) comprises a sequence encoding a detectable molecule. A detectable molecule is a molecule that can be visualized (e.g., using a naked eye, under a microscope, or using a light detection device such as a camera). In some embodiments, the detectable molecule is a fluorescent molecule, a bioluminescent molecule, or a molecule that provides color (e.g., β-galactosidase, β-lactamase, β-glucuronidase, or spheroidenone). In some embodiments, the detectable molecule is a fluorescent, bioluminescent or enzymatic protein or functional peptide or polypeptide thereof.

[0200] In some embodiments, fluorescent protein is a blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, a red fluorescent protein, or a functional peptide or polypeptide thereof.

[0201] In some embodiments, a detectable molecule is a bioluminescent protein or a functional peptide or polypeptide thereof. Non-limiting examples of bioluminescent proteins are firefly luciferase, click-beetle luciferase, and Renilla luciferase.

[0202] In some embodiments, a detectable molecule may be any polypeptide or protein that can be detected using methods known in the art. Non-limiting methods of detection are fluorescence imaging, luminescent imaging, bright field imaging, and include imaging facilitated by immunofluorescence or immunohistochemical staining. Viral Particles

[0203] Provided herein are viral particles e.g., AAV particles that comprise any of the nucleic acids (e.g., recombinant viral genomes) described herein. An AAV particle is a supramolecular assembly of 60 individual capsid protein subunits forming a non-enveloped T- 1 icosahedral lattice capable of protecting a single-stranded DNA genome. A mature AAV particle is approximately 20 nm in diameter, and its capsid is formed from three structural capsid proteins VP1, VP2, and VP3, with molecular masses of 87, 73, and 62 kDa, respectively, in a ratio of approximately 1:1:18. The 60 capsid proteins are arranged in an anti- parallel β-strand barreloid arrangement, resulting in a defined tropism and a high resistance to degradation.

[0204] In some embodiments, an AAV particle comprises a capsid encapsidating a nucleic acid (e.g., a recombinant AAV genomes comprising a nucleic acid comprising a least one miRNA binding nucleotide sequence). In some embodiments, an AAV particle described herein comprises a capsid protein. In some embodiments, an AAV particle described herein comprises a capsid protein comprising a wild-type AAV capsid protein sequence. For example, an AAV particle described herein comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or a MyoAAV, e.g., a MyoAAV2 or a MyoAAV9 capsid protein. In some embodiments, an AAV particle comprises a capsid protein of an AAV serotype 6, 8, 9, rh74, or MyoAAV.

[0205] In some embodiments, an AAV particle described herein comprises a capsid protein comprising one or more mutations, e.g., one or more amino acid substitutions.

[0206] In some embodiments, an AAV particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence comprises an AAV9 capsid protein. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence is encapsidated in an AAV9 capsid.

[0207] In some embodiments, an AAV particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence comprises an MyoAAV capsid protein, e.g., a MyoAAV2 or a MyoAAV9 capsid protein. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence is encapsidated in an MyoAAV capsid, e.g., a MyoAAV2 or a MyoAAV9 capsid protein.

[0208] In some embodiments, an AAV particle described herein comprises a capsid protein comprising a derivative of an AAV capsid protein or a hybrid AAV capsid comprising capsid proteins from more than one AAV serotype. Non-limiting examples of derivatives and pseudotypes include AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives / pseudotypes, and methods of producing such derivatives / pseudotypes are known in the art (see, e.g., Mol. Ther.2012 Apr; 20(4):699-708. The AAV vector toolkit: poised at the clinical crossroads. Asokan A, Schaffer DV, Samulski RJ.)

[0209] In some embodiments, an AAV particle described herein is replicative. A replicative AAV particle is capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture). In some embodiments, an AAV particle described herein is non-replicating. A non-replicating AAV particle is not capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture) but can infect the host and incorporate a nucleic acid described herein into the host’s genome for expression. In some embodiments, an AAV particle described herein is capable of infecting a host cell. In some embodiments, an AAV particle described herein is capable of facilitating stable integration of a nucleic acid described herein into the genome of a host cell, e.g., into a safe harbor locationsuch as AAVS1. In some embodiments, an AAV particle described herein is not capable of facilitating integration of a nucleic acid described herein into the genome of a host cell.

[0210] In some embodiments, an AAV particle described herein comprises a nucleic acid (e.g., a recombinant AAV genome) described herein. In some embodiments, a nucleic acid (e.g., a recombinant AAV genome) comprises two ITRs adjacent to the ends of a sequence comprising at least one miRNA binding nucleotide sequence. In some embodiments, the nucleic acid (e.g., recombinant AAV genome) is a single-stranded DNA vector. In some embodiments, an AAV particle described herein comprises two complementary DNA strands, forming a self-complementary AAV (scAAV). Pharmaceutical Compositions

[0211] Provided herein are compositions (e.g., pharmaceutical compositions) comprising a nucleic acid described herein (e.g., a nucleic acid comprising at least one miRNA binding nucleotide sequence), a recombinant viral genome described herein (e.g., a recombinant AAV genome) (e.g., a recombinant viral genome comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence), or a viral particle described herein (e.g., a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle)). In some aspects, the composition further comprises an excipient. In some aspects, the excipient is a pharmaceutically acceptable excipient. The terms carrier and excipient are used interchangeably.

[0212] In some embodiments, a carrier comprises a pharmaceutically-acceptable carrier. The term “carrier,” as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome)comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle) is combined or administered to a subject in need thereof. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Non-limiting examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose,polyacrylic acids, lubricating agents (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying agents, suspending agents, preserving agents (such as methyl-, ethyl-, and propyl-hydroxy-benzoates), and pH adjusting agents (such as inorganic and organic acids and bases), and solutions or compositions thereof. Other examples of carriers include phosphate buffered saline, HEPES-buffered saline, and water for injection, any of which may be optionally combined with one or more of calcium chloride dihydrate, disodium phosphate anhydrous, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose. Other examples of carriers that might be used include saline (e.g., sterilized, pyrogen-free saline), saline buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, mannitol, sorbitol, and glycerol. USP grade carriers are particularly useful for delivery of a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome)comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle) to a human subject.

[0213] Typically, such compositions may contain at least about 0.1%, about 0.1% to about 1%, about 1% to about 1.5%, about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 3.5%, about 3.5% to about 4%, about 4% to about 4.5%, about 4.5% to about 5%, about 5% to about 5.5%, about 5.5% to about 6%, about 6% to about 6.5%, about 6.5% to about 7%, about 7% to about 7.5%, or about 7.5% to about 8%, about 8% to about 8.5%, about 8.5% to about 9%, about 9% to about 9.5%, or about 9.5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, or about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 96% to about 98% of the nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome)comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or more, although the percentage of the active ingredient(s) may be varied. In some embodiments, a composition may contain a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAVgenome)comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle) at an amount of between about 1% or 2% and about 70% or 80% or more of the weight or volume of the total formulation. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome)comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle) may comprise between about 1% and about 90% of the weight or volume of a composition, e.g., a pharmaceutical composition, or about 2% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, or about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 96% to about 98% of the weight or volume of a composition, e.g., a pharmaceutical composition.

[0214] Naturally, amounts of therapeutic agent(s) (e.g., a nucleic acid, a recombinant AAV genome, or an AAV particle) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be designed.

[0215] Pharmaceutical compositions can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the provided nucleic acid, vector, or viral particle (“active ingredient”) into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0216] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0217] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the pharmaceutical composition is to be administered. The pharmaceutical composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0218] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the pharmaceutical composition.

[0219] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

[0220] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0221] Exemplary surface active agents and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitanfatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween®20), polyoxyethylene sorbitan (Tween®60), polyoxyethylene sorbitan monooleate (Tween®80), sorbitan monopalmitate (Span®40), sorbitan monostearate (Span®60), sorbitan tristearate (Span®65), glyceryl monooleate, sorbitan monooleate (Span®80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj®45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij®30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic®F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0222] Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0223] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0224] Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0225] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride,benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0226] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0227] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0228] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0229] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant®Plus, Phenonip®, methylparaben, Germall®115, Germaben®II, Neolone®, Kathon®, and Euxyl®.

[0230] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D- gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer’s solution, ethyl alcohol, and mixtures thereof.

[0231] Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0232] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, eveningprimrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0233] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral pharmaceutical compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0234] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di- glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0235] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solidpharmaceutical compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0236] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

[0237] Pharmaceutical compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0238] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0239] In some embodiments, the pharmaceutical compositions are suitable for sustained release and / or prolonged release. In some embodiments, the pharmaceutical compositions are suitable for sustained release and / or prolonged release over between 1 and 8 hours, between 8 and 24 hours, between 1 and 3 days, between 3 and 7 days, between 1 and 4 weeks, between 1 and 3 months, or between 3 and 12 months.

[0240] Solid pharmaceutical compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well ashigh molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a pharmaceutical composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating pharmaceutical compositions which can be used include polymeric substances and waxes. Solid pharmaceutical compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0241] The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a pharmaceutical composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

[0242] Dosage forms for topical and / or transdermal administration of a provided nucleic acid, vector, or viral particle may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and / or any needed preservatives and / or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches (e.g., microneedle transdermal patches), which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and / or by dispersing the active ingredient in a polymer matrix and / or gel.

[0243] Suitable devices for use in delivering intradermal pharmaceutical compositions include short needle devices. Intradermal pharmaceutical compositions can be administered bydevices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and / or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder delivery devices which use compressed gas to accelerate the provided nucleic acid, vector, or viral particle in powder form through the outer layers of the skin to the dermis are suitable.

[0244] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations such as liniments, lotions, oil-in-water and / or water-in- oil emulsions such as creams, ointments, and / or pastes, and / or solutions and / or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients.

[0245] Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally, the propellant may constitute 50 to 99.9% (w / w) of the pharmaceutical composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the pharmaceutical composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and / or solid anionic surfactant and / or a solid diluent.

[0246] Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations can be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and / or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and / or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

[0247] A pharmaceutical composition can be prepared, packaged, and / or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w / w) solution and / or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and / or one or more other of the additional ingredients. Otherophthalmically -administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are also contemplated as being within the scope of this disclosure.

[0248] Although the descriptions of the pharmaceutical compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such pharmaceutical compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the pharmaceutical compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with ordinary experimentation.

[0249] The provided nucleic acids, vectors, and viral particles are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the pharmaceutical compositions will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific pharmaceutical composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0250] The provided nucleic acids, vectors, viral particles, and pharmaceutical compositions can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, hydrogel (e.g., implanted hydrogel), biodegradable implants (e.g., biodegradable implants in muscular tissues, e.g., biodegradable implants in skeletal muscular tissues), and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and / or lymph supply, and / or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., itsstability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the provided nucleic acid, vector, viral particle, or pharmaceutical composition is suitable for topical administration to the eye of a subject.

[0251] The exact amount of a provided nucleic acid, vector, or viral particle required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular nucleic acid, vector, or viral particle, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses of the multiple doses include different or substantially the same amounts of a provided nucleic acid, vector, or viral particle. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) includes independently between 0.1 µg and 1 µg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a provided nucleic acid, vector, or viral particle. In certain embodiments, a dose includes independently between 1 mg and 3 mg, inclusive, of a provided nucleic acid, vector, or viral particle. In certain embodiments, a dose includes independently between 3 mg and 10 mg, inclusive, of a provided nucleic acid, vector, or viral particle. In certain embodiments, a dose includes independently between 10 mg and 30 mg, inclusive, of a provided nucleic acid, vector, or viral particle. In certain embodiments, a dose includes independently between 30 mg and 100 mg, inclusive, of a provided nucleic acid, vector, or viral particle.

[0252] Dose ranges as provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. Methods of Use

[0253] Provided are methods of contacting a cell with a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., an AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition).

[0254] In some embodiments, a method of contacting a cell may comprise, for example, contacting a cell in a culture with a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., an AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition).

[0255] In some embodiments, contacting a cell comprises adding a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., an AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) to a supernatant of a cell culture (e.g., a cell culture on a tissue culture plate or dish). In some embodiments, contacting comprises mixing a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinantAAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) with a cell culture (e.g., a suspension cell culture). In some embodiments, contacting a cell comprises mixing a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) with another solution, such as a cell culture media, and incubating a cell with the mixture.

[0256] In some embodiments, the concentration in a composition of AAV particles comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence may be ranging from about 103to about 1015particles / ml or about 106to about 1014particles / ml, or any values therebetween, such as, e.g., about 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014particles / ml.

[0257] In some embodiments, contacting a cell with a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) comprises administering a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) to a subject or device in which the cell is located. In some embodiments, contacting a cell comprises administering a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) directly to a cell, or into or substantially adjacent to a tissue of a subject in which the cell is present.

[0258] In some embodiments, a cell described herein is a cell isolated or derived from a subject. In some embodiments, a cell is a mammalian cell (e.g., a cell isolated or derived froma mammal). In some embodiments, a cell is a human cell. In some embodiments, a cell is isolated or derived from a particular tissue of a subject, such as muscle tissue. In some embodiments, a cell is a muscle cell. In some embodiments, a cell is a skeletal muscle cell. In some embodiments, a cell is a smooth muscle cell. In some embodiments, a cell is a cardiac muscle cell. In some embodiments, a cell is in vitro. In some embodiments, a cell is ex vivo. In some embodiments, a cell is in vivo, e.g., a cell is within a subject (e.g., within a tissue or organ of a subject). In some embodiments, a cell is a primary cell. In some embodiments, a cell is from a cell line (e.g., an immortalized cell line). In some embodiments a cell is a cancer cell or an immortalized cell. Methods of Treatment

[0259] Provided are methods of treating a metabolic disease comprising administering an effective amount of: a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) to a subject having the condition.

[0260] The terms “subject” and “patient,” as used herein, refer to organisms that are preferably mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like). In certain embodiments, the subject isa human (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle–aged adult, or senior adult)). Subjects may be non-human animals, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). In certain embodiments, the subject is a mammal. The subject may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.

[0261] The terms “treat,” “treating,” and “treatment” include any effect, e.g., lessening, reducing, modulating, ameliorating, or eliminating, that results in the improvement of a condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0262] In some embodiments, “administering” or “administration” means providing a material to a subject in need thereof in a manner that is pharmacologically useful.

[0263] In some embodiments, provided are methods of increasing MG29 expression in a cell.

[0264] In some embodiments, provided are methods of increasing glucose uptake in a cell of a subject. The phrase “improving glucose uptake into a skeletal muscle,” as used herein refers to an increase in Glut4-mediated glucose transport into a cell, e.g., a skeletal muscle cell.

[0265] In some embodiments, provided are methods of increasing insulin sensitivity in a subject in need thereof. The phrase “increasing insulin sensitivity,” as used herein, refers to increasing a subject’s responsiveness to insulin, e.g., a subject’s decrease in blood glucose levels following an increase in blood insulin levels.

[0266] In some embodiments, provided are methods of reducing glucose in a subject. The phrase “reducing glucose,” as used herein, refers to a decrease in blood glucose levels in a subject, which decrease includes a decrease in basal blood glucose levels (e.g., fasting blood glucose levels) and postprandial blood glucose levels.

[0267] In some embodiments, provided are methods of treating a metabolic disease in a subject in need thereof.

[0268] The term “metabolic disease,” as used herein, refers to a negative alteration of a subject’s processing and distribution of macronutrients including, e.g., proteins, fats, and carbohydrates.

[0269] In certain embodiments, the metabolic disease involves dysregulation of glucose homeostasis. In some embodiments, the metabolic disease is abdominal obesity-metabolic syndrome 3, abdominal obesity-metabolic syndrome 4, acquired metabolic disease, adenine phosphoribosyltransferase deficiency, amino acid metabolism disorder, amyloidosis, apparent mineralocorticoid excess syndrome, body fluid hypertonicity, carbohydrate metabolism disorder, catabolic disorder, DNA repair-deficiency disorder, electrolyte metabolism disorder, exercise intolerance, fatty acid oxidation disorder, GAND syndrome, gamma-amino butyric acid metabolism disorder, glutaric acidemia II, glycerol kinase deficiency, hyalinosis, hyperchylomicronemia, hyperlactatemia, hypogonadotropism, hypomyelination, inherited metabolic disorder, insulin resistance, insulinopathy, ketosis, lipid metabolism disorder, lysosomal acid lipase deficiency, lysosomal storage disease, MOMO syndrome, metabolic acidosis, metabolic encephalopathy, metabolic myopathy, metabolic reprogramming, metabolic syndrome X, mineral metabolism disorder, mitochondrial disease, peroxisomal disorder, protein metabolism disorder, refeeding syndrome, ribosomopathy, severe combined immunodeficiency, severe dermatitis, multiple allergies and metabolic wasting syndrome, thyroid hormone resistance syndrome, tumor lysis syndrome, or vitamin metabolism disorder.In some embodiments, the metabolic disease is diabetes. In some embodiments, the metabolic disease is type II diabetes. In some embodiments, the metabolic disease is type I diabetes. In certain embodiments, the metabolic disease is gestational diabetes. In certain embodiments, the metabolic disease is hyperglycemia. In certain embodiments, the metabolic disease is obesity.

[0270] In some embodiments, a method comprises administering an effective amount of a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) to a subject in need thereof. The term “effective amount,” as used herein, refers to the amount of nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) sufficient to effect beneficial or desired results, e.g., improving glucose uptake into a skeletal muscle cell. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. The term effective amount can be considered to include therapeutically and / or prophylactically effective amounts of a compound.

[0271] In some embodiments, a method comprises administering a therapeutically effective amount of a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) to a subject. The term “therapeutically effective amount,” as used herein, refers to an amount of a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition), which is effective for producing a desired therapeutic effect, e.g., an increase in glucose uptake into skeletal muscle in at least a sub-population of cells in amammal, e.g., a human, or a subject (e.g., a human subject) at a reasonable benefit / risk ratio applicable to any medical treatment.

[0272] The term “prophylactically effective amount,” as used herein, refers to an amount of nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition), which is effective for producing a desired prophylactic effect in at least a sub-population of cells in a mammal, e.g., a human by reducing, minimizing or eliminating the risk of developing a condition, e.g., a metabolic disease or by reducing or minimizing severity of a condition, e.g., a metabolic disease at a reasonable benefit / risk ratio applicable to any medical treatment.

[0273] In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) is administered at an effective amount by systemic administration, e.g., parenterally. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) is administered at an effective amount to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro- ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs. In some embodiments, the step of administering or contacting comprises parenteral administration. In some embodiments, the step of administering or contacting comprises intramuscular administration. In some embodiments, the step of administering or contacting comprises intramuscular injection. In some embodiments, the step of administering or contacting comprises injection into a skeletal muscle. In some embodiments, the step of administering or contacting comprises topical administration. In some embodiments, the step of administering or contacting comprises transdermal administration.

[0274] In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) is administered at an effective amount to a subject once. In some embodiments, a nucleic acid comprising at least one miRNA binding nucleotide sequence, a recombinant viral genome (e.g., a recombinant AAV genome) comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence, a viral particle comprising a nucleic acid comprising at least one miRNA binding nucleotide sequence (e.g., an AAV particle), or a composition (e.g., a pharmaceutical composition) is administered at an effective amount to a subject multiple times (e.g., twice, three times, four times, five times, six times, or more). Repeated administration to a subject may be conducted at a regular interval (e.g., daily, every other day, twice per week, weekly, twice per month, monthly, every six months, once per year, or less or more frequently) as necessary to treat (e.g., improve or alleviate) one or more symptoms of a disease, disorder, or condition, e.g., a metabolic disease in the subject. EXAMPLES Example 1.

[0275] Over the past decade, microRNAs (miRNAs) have emerged as potential therapeutic tools in the fields of cancer, metabolic diseases, and aging. Gene therapy is a widely explored approach that aims to modify specific gene targets and their expression patterns to manipulate an organism's physiology or disease underpinnings. miRNAs play a crucial role in RNA regulation and the subsequent control of protein expression.

[0276] MG29 is a gene encoding a protein with essential roles in control of calcium signaling and glucose uptake into skeletal muscle. MG29 protein is predominantly expressed in skeletal muscle and is undetectable in heart, liver, kidney, and brain.

[0277] Mice with ablation of MG29 developed glucose tolerance and insulin-resistance, resembling T2D.

[0278] Sequence analyses revealed that the protein for MG29 is well conserved between rodents and humans with greater than 94% identity. Compared with other human genes, the MG29 gene contains an unusually long 3′UTR. It was found that unwinding of the 3′UTR could enhance MG29 protein expression. In the case of MG29, it was unexpectedly found that miRNAs bound to the 3′UTR of MG29 RNA to inhibit its translation. Database mining usingTargetScan identified multiple miRNA-binding sites in the MG293′UTR, which could contribute to posttranscriptional control of MG29 protein expression (FIG.1).

[0279] Different binding sequences were screened for miRNAs for their potential to compete with the 3′ UTR, and therefore enhance the translation of the mRNA into MG29 protein. Ultimately, a combination of the multiple miRNA-sponge sequences was found to be most effective in boosting MG29 protein expression.

[0280] It was previously demonstrated that MG29 plays a vital role in the biogenesis of the transverse tubule (TT) network, as a MG29 knockout in mice led to disruption of the TT network and calcium signaling where Glut4-mediated glucose uptake takes place. The absence of MG29 disrupted the TT membrane network in skeletal muscle of mice and led to decreased ability of insulin-mediated uptake of blood glucose into skeletal muscle, leading to glucose intolerance in MG29- / -mice (FIG.2A). In addition, the MG29- / -mice displayed reduced sensitivity to glucose take up in response to insulin stimulation (FIG.2B). Similar results were previous disclosed in the patent (US Patent 8,603,993, issued December 10, 2013, refer to Figure 5C and 6). However, overexpression of MG29 using AAV gene delivery improved glucose-handling in mice (FIG.2C).

[0281] Exercise induced acute elevation of MG29 protein in skeletal muscle. Compared with the basal level of MG29, after two hour of exercise training of wild type mice, MG29 protein was measurable increased, whereas the expression level of MG53 (a muscle specific- protein) remained unchanged following exercise (FIG.3).

[0282] The coding sequence (CDS) of MG29 could be expressed in C2C12 cells without its 3′ UTR (FIG.4A). However, with the 3′ UTR present in the plasmid (full-length, FL), MG29 protein expression was severely suppressed; this was observed both in the mouse and human MG29 gene (FIG.4A). Transient overexpression of the 3′ UTR in C2C12 cells uncovered endogenous MG29 protein expression in a dose-dependent manner (FIG.4B). Importantly, miR-Sponges that contained a combination of multiple miR-binding sites, e.g., mir-29-sponge (sequence shown below), were required to effectively boost MG29 expression in muscle cells (FIG.5B).

[0283] Sequences for pSiCHECK2 constructs are shown below: miR-29-sponge: 5’- GCAGCCCGATGCTATTGGTGCTTaattGCAGCCCGATGCTATTGGTGCTTaattGCAGCC CGATGCTATTGGTGCTT (SEQ ID NO: 8); 5’GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAG GCCGATGCTATTGGTGCTT (SEQ ID NO: 9); BS-miR-29: 5’- GCAGCCCGATGCTATTGGTGCTT (SEQ ID NO: 5); BS-miR-375: 5’-CTCCAGAGGTACAAAGAACAAAA (SEQ ID NO: 6); and BS-miR-29-mut: 5’ GCAGCCCGATGCTATACCACGAT (SEQ ID NO: 10).

[0284] Based on these findings, a therapeutic approach was developed that employed AAV delivery of the 3′ UTR or a “miR-sponge” to increase MG29 expression in skeletal muscle as a novel therapeutic approach to directly treat hyperglycemia associated with diabetes. Sequences used in the miR-sponges include the sequences below. Position 1244-1250 of SYPL23’ UTR (hsa-miR-29a-3p) 5’-GCAGGCCGAUGCUAUUGGUGCUU (SEQ ID NO: 1) Position 1488-1494 of human MG293’UTR (hsa-miR-181a-5p) 5’-TAAGCATCCTAAAGGTGAATGTG (SEQ ID NO: 2) Position 1857-1863 of human MG293’UTR (hsa-miR-375) 5’-CTCCAGAGACACAGAGAACAAAG (SEQ ID NO: 3) Position 2533-2539 of SYPL23'UTR (hsa-miR-29a2-39 5’-UUGCAUGUUGUUUUCUGGUGCUU (SEQ ID NO: 4) Position 886-892 of mouse MG293’UTR (mmu-miR-29a-3p) 5’-GCAGCCCGATGCTATTGGTGCTT (SEQ ID NO: 5) Position 1533-1539 of mouse MG293’UTR (mmu-miR-375-3p) 5’-CTCCAGAGGTACAAAGAACAAAA (SEQ ID NO: 6) Position 2181-2187 of mouse MG293’UTR (mmu-miR-29a-3p) 5’-TTGCATGTTGTTCTGTGGTGCTT (SEQ ID NO: 7) MSSTESPGRTSDKSPQQVDRLLLGLRWQRLEEPLGFIKVLQWLFAIFAFGSCGSYSGET GALVLCNNEAKDVSSIIVLFGYPFRLYQVQYEMPLCDQDSTSKTMNLMGDFSAPAEFF VTLGIFSFFYTMAALVIYLRFHKLYTENKRFPLVDFCVTVSFTFFWLVAAAAWGKGLT DVKGATRPSSLTAAMSVCHGEEAVCSAGATPSMGLANLSVLFGFINFFLWAGNCWFV FKETPWHGQGQDQGQGPSQESAAEQGAVEKQ Mouse MG29 (SEQ ID NO: 60) MSSTESAGRTADKSPRQQVDRLLVGLRWRRLEEPLGFIKVLQWLFAIFAFGSCGSYSG ETGAMVRCNNEAKDVSSIIVAFGYPFRLHRIQYEMPLCDEESSSKTMHLMGDFSAPAE FFVTLGIFSFFYTMAALVIYLRFHNLYTENKRFPLVDFCVTVSFTFFWLVAAAAWGKG LTDVKGATRPSSLTAAMSVCHGEEAVCSAGATPSMGLANISVLFGFINFFLWAGNCW FVFKETPWHGQGQGQDQDQDQDQGQGPSQESAAEQGAVEKQ Human MG29 (SEQ ID NO: 61)

[0285] Sequences for nucleic acids containing single miRNA binding sequences and multiple copies of single miRNA binding sequences are shown below: 1. miR29a1-up5’GATCTGGATCCGCAGCCCGATGCTATTGGTGCTTaattGCAGCCCGATGCTATTGGT GCTTaattGCAGCCCGATGCTATTGGTGCTTG (SEQ ID NO: 11) miR-29a1-dn 5’AATTCAAGCACCAATAGCATCGGGCTGCaattAAGCACCAATAGCATCGGGCTGCa attAAGCACCAATAGCATCGGGCTGCGGATCCA (SEQ ID NO: 12) 2. miR29a2-up 5’GATCTGGATCCTTGCATGTTGTTCTGTGGTGCTTaattTTGCATGTTGTTCTGTGGTG CTTaattTTGCATGTTGTTCTGTGGTGCTTG (SEQ ID NO: 13) miR29a2-dn 5’AATTCAAGCACCACAGAACAACATGCAAaattAAGCACCACAGAACAACATGCAA aattAAGCACCACAGAACAACATGCAAGGATCCA (SEQ ID NO: 14) 3. miR375-up 5’GATCTGGATCCCTCCAGAGGTACAAAGAACAAAAaattCTCCAGAGGTACAAAGA ACAAAAaattCTCCAGAGGTACAAAGAACAAAAG (SEQ ID NO: 15) miR375-dn 5’AATTCTTTTGTTCTTTGTACCTCTGGAGaattTTTTGTTCTTTGTACCTCTGGAGaattT TTTGTTCTTTGTACCTCTGGAGGGATCCA (SEQ ID NO: 16) 4. miR181-up 5’GATCTGGATCCTTAGCACCCCAAAGATGAATGTAaattTTAGCACCCCAAAGATGA ATGTAaattTTAGCACCCCAAAGATGAATGTAG (SEQ ID NO: 17) miR181-dn 5’AATTCTACATTCATCTTTGGGGTGCTAAaattTACATTCATCTTTGGGGTGCTAAaatt TACATTCATCTTTGGGGTGCTAAGGATCCA (SEQ ID NO: 18) miR29a1-UP 5’GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAG GCCGATGCTATTGGTGCTT (SEQ ID NO: 9) miR29a1-DN 5’AAGCACCAGAAAACAACATGCAAaattAAGCACCAGAAAACAACATGCAAaattAA GCACCAGAAAACAACATGCAA (SEQ ID NO: 19) 2. miR29a2-UP 5’TTGCATGTTGTTTTCTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTTaattTTGCAT GTTGTTTTCTGGTGCTT (SEQ ID NO: 20) miR29a2-DN5’AAGCACCAGAAAACAACATGCAAaattAAGCACCAGAAAACAACATGCAAaattAA GCACCAGAAAACAACATGCAA (SEQ ID NO: 19) 3. miR375-UP 5’CTCCAGAGACACAGAGAACAAAGaattCTCCAGAGACACAGAGAACAAAGaattCTC CAGAGACACAGAGAACAAAG (SEQ ID NO: 21) miR375-DN 5’CTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGT TCTCTGTGTCTCTGGAG (SEQ ID NO: 22) 4. miR181-UP 5’TAAGCATCCTAAGGATGAATGTGaattTAAGCATCCTAAGGATGAATGTGaattTAAG CATCCTAAGGATGAATGTG (SEQ ID NO: 23) miR181-DN 5’CACATTCATCCTTAGGATGCTTAaattCACATTCATCCTTAGGATGCTTAaattCACAT TCATCCTTAGGATGCTTA (SEQ ID NO: 24)

[0286] Sequences for nucleic acids containing two miRNA binding sequences and multiple copies of two miRNA binding sequences are shown below: miR29a1-375-UP 5’GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAG GCCGATGCTATTGGTGCTTaattCTCCAGAGACACAGAGAACAAAGaattCTCCAGAGA CACAGAGAACAAAGaattCTCCAGAGACACAGAGAACAAAG (SEQ ID NO: 25) miR29a1-375-DN 5’CTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGT TCTCTGTGTCTCTGGAGaattAAGCACCAATAGCATCGGCCTGCaattAAGCACCAATA GCATCGGCCTGCaattAAGCACCAATAGCATCGGCCTGC (SEQ ID NO: 26) 2. miR375-29a2-UP 5’CTCCAGAGACACAGAGAACAAAGaattCTCCAGAGACACAGAGAACAAAGaattCTC CAGAGACACAGAGAACAAAGaattTTGCATGTTGTTTTCTGGTGCTTaattTTGCATGTT GTTTTCTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTT (SEQ ID NO: 27) miR375-29a2-DN 5’AAGCACCAGAAAACAACATGCAAaattAAGCACCAGAAAACAACATGCAAaattAA GCACCAGAAAACAACATGCAAaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTC TCTGTGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAG (SEQ ID NO: 28) 3. miR29a1-29a2-UP5’GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAG GCCGATGCTATTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTTaattTTGCATGTTGT TTTCTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTT (SEQ ID NO: 29) miR-29a1-29a2-DN 5’AAGCACCAGAAAACAACATGCAAaattAAGCACCAGAAAACAACATGCAAaattAA GCACCAGAAAACAACATGCAAaattAAGCACCAATAGCATCGGCCTGCaattAAGCAC CAATAGCATCGGCCTGCaattAAGCACCAATAGCATCGGCCTGC (SEQ ID NO: 30) 1. miR29a1-375-up 5’GATCTGGATCCGCAGCCCGATGCTATTGGTGCTTaattCTCCAGAGGTACAAAGAA CAAAAaattGCAGCCCGATGCTATTGGTGCTTaattCTCCAGAGGTACAAAGAACAAAA aattGCAGCCCGATGCTATTGGTGCTTaattCTCCAGAGGTACAAAGAACAAAAG (SEQ ID NO: 31) miR29a1-375-dn 5’AATTCTTTTGTTCTTTGTACCTCTGGAGaattAAGCACCAATAGCATCGGGCTGCaatt TTTTGTTCTTTGTACCTCTGGAGaattAAGCACCAATAGCATCGGGCTGCaattTTTTGT TCTTTGTACCTCTGGAGaattAAGCACCAATAGCATCGGGCTGCGGATCCA (SEQ ID NO: 32) 2. miR375-29a2-up 5’CTCCAGAGGTACAAAGAACAAAAaattCTCCAGAGGTACAAAGAACAAAAaattCTC CAGAGGTACAAAGAACAAAAaattTTGCATGTTGTTCTGTGGTGCTTaattTTGCATGTT GTTCTGTGGTGCTTaattTTGCATGTTGTTCTGTGGTGCTT (SEQ ID NO: 33) miR375-29a2-dn 5’AAGCACCACAGAACAACATGCAAaattAAGCACCACAGAACAACATGCAAaattAAG CACCACAGAACAACATGCAAaattTTTTGTTCTTTGTACCTCTGGAGaattTTTTGTTCTT TGTACCTCTGGAGaattTTTTGTTCTTTGTACCTCTGGAG (SEQ ID NO: 34) 3. miR29a1-29a2-up 5’GATCTGGATCCGCAGCCCGATGCTATTGGTGCTTaattTTGCATGTTGTTCTGTGGT GCTTaattGCAGCCCGATGCTATTGGTGCTTaattTTGCATGTTGTTCTGTGGTGCTTaatt GCAGCCCGATGCTATTGGTGCTTaattTTGCATGTTGTTCTGTGGTGCTTG (SEQ ID NO: 35) miR29a1-29a2-dn 5’AATTCAAGCACCACAGAACAACATGCAAaattAAGCACCAATAGCATCGGGCTGCa attAAGCACCACAGAACAACATGCAAaattAAGCACCAATAGCATCGGGCTGCaattAAGCACCACAGAACAACATGCAAaattAAGCACCAATAGCATCGGGCTGCGGATCCA (SEQ ID NO: 36) 4. miR-181a-375-up 5’GATCTGGATCCTTAGCACCCCAAAGATGAATGTATTAGCACCCCAAAGATGAAT GTAaattTTAGCACCCCAAAGATGAATGTAaattCTCCAGAGGTACAAAGAACAAAAaat tCTCCAGAGGTACAAAGAACAAAAaattCTCCAGAGGTACAAAGAACAAAAG (SEQ ID NO: 37) miR-181a-375-dn 5’AATTCTTTTGTTCTTTGTACCTCTGGAGaattTTTTGTTCTTTGTACCTCTGGAGaattT TTTGTTCTTTGTACCTCTGGAGaattTACATTCATCTTTGGGGTGCTAAaattTACATTCA TCTTTGGGGTGCTAATACATTCATCTTTGGGGTGCTAAGGATCCA (SEQ ID NO: 38) 5. miR-29a1-181a-up 5’GATCTGGATCCGCAGCCCGATGCTATTGGTGCTTaattGCAGCCCGATGCTATTGGT GCTTaattGCAGCCCGATGCTATTGGTGCTTaattTTAGCACCCCAAAGATGAATGTAaat tTTAGCACCCCAAAGATGAATGTAaattTTAGCACCCCAAAGATGAATGTAG (SEQ ID NO: 39) miR-29a1-181a-dn 5’AATTCTACATTCATCTTTGGGGTGCTAAaattTACATTCATCTTTGGGGTGCTAAaatt TACATTCATCTTTGGGGTGCTAAaattAAGCACCAATAGCATCGGGCTGCaattAAGCA CCAATAGCATCGGGCTGCaattAAGCACCAATAGCATCGGGCTGCGGATCCA (SEQ ID NO: 40) 4. miR181-375-UP 5’TAAGCATCCTAAGGATGAATGTGaattTAAGCATCCTAAGGATGAATGTGaattTAAG CATCCTAAGGATGAATGTGaattCTCCAGAGACACAGAGAACAAAGaattCTCCAGAG ACACAGAGAACAAAGaattCTCCAGAGACACAGAGAACAAAG (SEQ ID NO: 41) miR181-375-DN 5’CTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGT TCTCTGTGTCTCTGGAGaattCACATTCATCCTTAGGATGCTTAaattCACATTCATCCTT AGGATGCTTAaattCACATTCATCCTTAGGATGCTTA (SEQ ID NO: 42) 5. miR29a1-181-UP 5’GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAG GCCGATGCTATTGGTGCTTaattTAAGCATCCTAAGGATGAATGTGaattTAAGCATCCT AAGGATGAATGTGaattTAAGCATCCTAAGGATGAATGTG (SEQ ID NO: 43) miR29a1-181-DN5’CACATTCATCCTTAGGATGCTTAaattCACATTCATCCTTAGGATGCTTAaattCACAT TCATCCTTAGGATGCTTAaattAAGCACCAATAGCATCGGCCTGCaattAAGCACCAAT AGCATCGGCCTGCaattAAGCACCAATAGCATCGGCCTGC (SEQ ID NO: 44) 6. miR181-29a2-UP 5′TAAGCATCCTAAGGATGAATGTGaattTAAGCATCCTAAGGATGAATGTGaattTAAG CATCCTAAGGATGAATGTGaattTTGCATGTTGTTTTCTGGTGCTTaattTTGCATGTTGT TTTCTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTT (SEQ ID NO: 62) miR181-29a2-DN 5′CACATTCATCCTTAGGATGCTTAaattCACATTCATCCTTAGGATGCTTAaattCACAT TCATCCTTAGGATGCTTAaattAAGCACCAGAAAACAACATGCAAaattAAGCACCAGA AAACAACATGCAAaattAAGCACCAGAAAACAACATGCAA (SEQ ID NO: 63)

[0287] Sequences for nucleic acids containing three miRNA binding sequences and multiple copies of three miRNA binding sequences are shown below: 1. miR-29a1-181a-375-up 5’GATCTGGATCCGCAGCCCGATGCTATTGGTGCTTaattGCAGCCCGATGCTATTGGT GCTTaattGCAGCCCGATGCTATTGGTGCTTaattTTAGCACCCCAAAGATGAATGTAaat tTTAGCACCCCAAAGATGAATGTAaattTTAGCACCCCAAAGATGAATGTAaattCTCCA GAGGTACAAAGAACAAAAaattCTCCAGAGGTACAAAGAACAAAAaattCTCCAGAGG TACAAAGAACAAAAG (SEQ ID NO: 45) miR-29a1-181a-375-dn 5’AATTCTTTTGTTCTTTGTACCTCTGGAGaattTTTTGTTCTTTGTACCTCTGGAGaattT TTTGTTCTTTGTACCTCTGGAGaattTACATTCATCTTTGGGGTGCTAAaattTACATTCA TCTTTGGGGTGCTAAaattTACATTCATCTTTGGGGTGCTAAaattAAGCACCAATAGCA TCGGGCTGCaattAAGCACCAATAGCATCGGGCTGCaattAAGCACCAATAGCATCGGG CTGCGGATCCA (SEQ ID NO: 46) 2. miR29a1-375-29a2-up 5’GATCTGGATCCGCAGCCCGATGCTATTGGTGCTTaattCTCCAGAGGTACAAAGAA CAAAAaattTTGCATGTTGTTCTGTGGTGCTTaattGCAGCCCGATGCTATTGGTGCTTaa ttCTCCAGAGGTACAAAGAACAAAAaattTTGCATGTTGTTCTGTGGTGCTTaattGCAGC CCGATGCTATTGGTGCTTaattCTCCAGAGGTACAAAGAACAAAAaattTTGCATGTTG TTCTGTGGTGCTTG (SEQ ID NO: 47) miR29a1-375-29a2-dn 5’AATTCAAGCACCACAGAACAACATGCAAaattTTTTGTTCTTTGTACCTCTGGAGaat tAAGCACCAATAGCATCGGGCTGCaattAAGCACCACAGAACAACATGCAAaattTTTTGTTCTTTGTACCTCTGGAGaattAAGCACCAATAGCATCGGGCTGCaattAAGCACCAC AGAACAACATGCAAaattTTTTGTTCTTTGTACCTCTGGAGaattAAGCACCAATAGCAT CGGGCTGCGGATCCA (SEQ ID NO: 48) 1. miR29a1-181-375-UP 5’GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAG GCCGATGCTATTGGTGCTTaattTAAGCATCCTAAGGATGAATGTGaattTAAGCATCCT AAGGATGAATGTGaattTAAGCATCCTAAGGATGAATGTGaattCTCCAGAGACACAGA GAACAAAGaattCTCCAGAGACACAGAGAACAAAGaattCTCCAGAGACACAGAGAAC AAAG (SEQ ID NO: 49) miR29a1-181-375-DN 5’CTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGT TCTCTGTGTCTCTGGAGaattCACATTCATCCTTAGGATGCTTAaattCACATTCATCCTT AGGATGCTTAaattCACATTCATCCTTAGGATGCTTAaattAAGCACCAATAGCATCGG CCTGCaattAAGCACCAATAGCATCGGCCTGCaattAAGCACCAATAGCATCGGCCTGC (SEQ ID NO: 50) 2. miR29a1-375-29a2-UP 5’GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAG GCCGATGCTATTGGTGCTTaattCTCCAGAGACACAGAGAACAAAGaattCTCCAGAGA CACAGAGAACAAAGaattCTCCAGAGACACAGAGAACAAAGaattTTGCATGTTGTTTT CTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTTaattTTGCATGTTGTTTTCTGGTGC TT (SEQ ID NO: 51) miR29a1-375-29a2-DN 5’AAGCACCAGAAAACAACATGCAAaattAAGCACCAGAAAACAACATGCAAaattAA GCACCAGAAAACAACATGCAAaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTC TCTGTGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAGaattAAGCACCAATAGCA TCGGCCTGCaattAAGCACCAATAGCATCGGCCTGCaattAAGCACCAATAGCATCGGC CTGC (SEQ ID NO: 52) 3. miR181-375-29a2-UP 5′TAAGCATCCTAAGGATGAATGTGaattTAAGCATCCTAAGGATGAATGTGaattTAAG CATCCTAAGGATGAATGTGaattCTCCAGAGACACAGAGAACAAAGaattCTCCAGAG ACACAGAGAACAAAGaattCTCCAGAGACACAGAGAACAAAGaattTTGCATGTTGTTT TCTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTTaattTTGCATGTTGTTTTCTGGTG CTT (SEQID NO: 64) miR181-375-29a2-DN5′CACATTCATCCTTAGGATGCTTAaattCACATTCATCCTTAGGATGCTTAaattCACAT TCATCCTTAGGATGCTTAaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTCTCTG TGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAGaattAAGCACCAGAAAACAACA TGCAAaattAAGCACCAGAAAACAACATGCAAaattAAGCACCAGAAAACAACATGCA A (SEQ ID NO: 65)

[0288] Sequences for nucleic acids containing four miRNA binding sequences and multiple copies of four miRNA binding sequences are shown below: 1. miR29a1-181-375-29a2-up 5’GATCCGCAGCCCGATGCTATTGGTGCTTaattGCAGCCCGATGCTATTGGTGCTTaatt GCAGCCCGATGCTATTGGTGCTTaattTTAGCACCCCAAAGATGAATGTAaattTTAGC ACCCCAAAGATGAATGTAaattTTAGCACCCCAAAGATGAATGTAGAATTCCTCCAG AGGTACAAAGAACAAAAaattCTCCAGAGGTACAAAGAACAAAAaattCTCCAGAGGT ACAAAGAACAAAAaattTTGCATGTTGTTCTGTGGTGCTTaattTTGCATGTTGTTCTGT GGTGCTTaattTTGCATGTTGTTCTGTGGTGCTTC (SEQ ID NO: 53) miR29a1-181-375-29a2-dn 5’TCGAGAAGCACCACAGAACAACATGCAAaattAAGCACCACAGAACAACATGCAA aattAAGCACCACAGAACAACATGCAAaattTTTTGTTCTTTGTACCTCTGGAGaattTTTT GTTCTTTGTACCTCTGGAGaattTTTTGTTCTTTGTACCTCTGGAGGAATTCTACATTC ATCTTTGGGGTGCTAAaattTACATTCATCTTTGGGGTGCTAAaattTACATTCATCTTT GGGGTGCTAAaattAAGCACCAATAGCATCGGGCTGCaattAAGCACCAATAGCATCG GGCTGCaattAAGCACCAATAGCATCGGGCTGCG(SEQ ID NO: 54) 1. miR29a1-181-375-29a2-UP GCAGGCCGATGCTATTGGTGCTTaattGCAGGCCGATGCTATTGGTGCTTaattGCAGG CCGATGCTATTGGTGCTTaattTAAGCATCCTAAGGATGAATGTGaattTAAGCATCCTA AGGATGAATGTGaattTAAGCATCCTAAGGATGAATGTGaattCTCCAGAGACACAGAG AACAAAGaattCTCCAGAGACACAGAGAACAAAGaattCTCCAGAGACACAGAGAACA AAGaattTTGCATGTTGTTTTCTGGTGCTTaattTTGCATGTTGTTTTCTGGTGCTTaattTT GCATGTTGTTTTCTGGTGCTT (SEQ ID NO: 55) miR29a1-181-375-29a2-DN AAGCACCAGAAAACAACATGCAAaattAAGCACCAGAAAACAACATGCAAaattAAGC ACCAGAAAACAACATGCAAaattCTTTGTTCTCTGTGTCTCTGGAGaattCTTTGTTCTC TGTGTCTCTGGAGaattCTTTGTTCTCTGTGTCTCTGGAGaattCACATTCATCCTTAGGA TGCTTAaattCACATTCATCCTTAGGATGCTTAaattCACATTCATCCTTAGGATGCTTAaattAAGCACCAATAGCATCGGCCTGCaattAAGCACCAATAGCATCGGCCTGCaattAAG CACCAATAGCATCGGCCTGC (SEQ ID NO: 56)

[0289] The sequence for eGFP is shown below: >eGFP sequences (720 bp) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGC TGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG ATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC CGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGC GCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAAC TTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCT GAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACA AGTAA (SEQ ID NO: 57)

[0290] The sequence NM_001359114.1:498-1751 Mus musculus solute carrier family 2 (facilitated glucosetransporter), member 4 (Slc2a4), transcript variant 2, mRNA (1254 bp) is shown below. >NM_001359114.1:498-1751 Mus musculus solute carrier family 2 (facilitated glucosetransporter), member 4 (Slc2a4), transcript variant 2, mRNA (1254 bp) ATGATCTCTTCCTTTCTCATTGGCATCATTTCTCAATGGTTGGGAAGGAAAAGGGC TATGCTGGCCAACAATGTCTTGGCCGTGTTGGGGGGCGCCCTCATGGGCCTAGCCA ATGCCGCGGCCTCCTATGAGATACTCATTCTTGGACGGTTCCTCATTGGCGCCTAC TCAGGGCTAACATCAGGGCTGGTGCCCATGTATGTGGGAGAAATCGCCCCCACTC ATCTTCGGGGTGCCTTGGGAACACTCAACCAACTGGCCATCGTCATTGGCATTCTG GTTGCCCAGGTGCTGGGCTTGGAGTCTATGCTGGGCACAGCTACCCTGTGGCCACT GCTTCTGGCTCTCACAGTACTCCCTGCTCTCCTGCAGCTGATTCTGCTGCCCTTCTG TCCTGAGAGCCCCAGATACCTCTACATCATCCGGAACCTGGAGGGGCCTGCCCGA AAGAGTCTAAAGCGCCTGACCGGCTGGGCTGATGTGTCTGACGCACTAGCTGAGC TGAAGGATGAGAAACGGAAGTTGGAGAGAGAGCGTCCAATGTCCTTGCTCCAGCTCCTGGGCAGCCGCACCCACCGGCAGCCTCTGATCATCGCAGTGGTGCTGCAGCTG AGCCAACAGCTCTCAGGCATCAATGCTGTTTTCTACTATTCAACCAGCATCTTCGA GTCGGCTGGGGTGGGACAGCCAGCCTACGCCACCATAGGAGCTGGTGTGGTCAAT ACGGTCTTCACGTTGGTCTCGGTGCTCTTAGTAGAACGAGCTGGACGACGGACACT CCATCTGTTGGGCCTGGCCGGCATGTGTGGCTGTGCCATCTTGATGACCGTGGCTC TGCTGCTGCTGGAACGGGTTCCAGCCATGAGCTATGTCTCCATCGTGGCCATATTT GGCTTTGTGGCCTTCTTTGAGATTGGCCCTGGCCCCATTCCCTGGTTCATTGTGGCA GAGCTCTTCAGCCAGGGCCCCCGCCCAGCCGCCATGGCTGTCGCTGGTTTCTCCAA CTGGACCTGTAACTTCATTGTCGGCATGGGTTTCCAGTATGTTGCGGATGCTATGG GTCCTTACGTCTTCCTTCTATTTGCCGTCCTCCTGCTTGGCTTCTTCATCTTCACCTT CCTAAAAGTGCCTGAAACCAGAGGCCGGACGTTTGACCAGATCTCAGCTGCCTTC CGACGGACACCTTCCCTTTTAGAGCAGGAGGTGAAACCCAGTACAGAACTTGAAT ACTTAGGGCCAGATGAGAATGACTGA (SEQ ID NO: 58)

[0291] The sequence of NM_001079932.3:107-1540 Mus musculus tripartite motif- containing 72 (Trim72), mRNA(1434 bp) is shown below: >NM_001079932.3:107-1540 Mus musculus tripartite motif-containing 72 (Trim72), mRNA(1434 bp) ATGTCGGCTGCACCCGGCCTTCTGCGTCAGGAACTGTCCTGCCCACTGTGCTTGCA GCTGTTCGATGCGCCAGTGACGGCTGAGTGTGGCCACAGTTTCTGCCGTGCCTGCC TGATCCGGGTGGCAGGGGAGCCTGCTGCGGACGGCACAGTTGCCTGTCCCTGTTGT CAGGCACCTACACGGCCGCAGGCTCTAAGCACTAACCTCCAGTTGTCACGCCTTGT GGAGGGTTTGGCGCAAGTGCCCCAAGGCCACTGCGAGGAACACCTGGATCCACTG AGCATCTACTGCGAGCAGGACCGCACACTTGTGTGTGGTGTGTGTGCCTCGCTCGG TTCTCACCGTGGTCATCGTCTCCTGCCTGCCGCTGAAGCCCAAGCACGCCTCAAGA CACAGCTTCCACAGCAGAAGATGCAGCTGCAGGAGGCATGCATGCGCAAGGAGA AGACTGTAGCGGTGCTGGAGCATCAGCTGGTGGAGGTGGAGGAGACAGTGCGCCA GTTCCGGGGAGCTGTCGGGGAGCAGCTGGGGAAGATGCGGATGTTCCTGGCTGCC CTAGAAAGTTCTCTGGACCGTGAAGCAGAAAGGGTTCGGGGTGATGCTGGGGTTG CCTTGCGTCGGGAGCTGTCAAGCCTGAACTCTTACCTAGAGCAACTGAGGCAGAT GGAGAAGGTGCTGGAGGAGGTGGCTGACAAGCCACAGACAGAATTCCTCATGAA ATTCTGCCTGGTAACCAGCAGGCTGCAGAAGATCCTGTCAGAGTCACCACCACCG GCAAGGCTAGATATCCAGCTGCCTGTCATCTCAGATGACTTCAAATTCCAGGTGTG GAAGAAGATGTTCCGGGCTCTGATGCCAGCGCTGGAGGAACTGACTTTTGACCCC AGCTCTGCGCACCCGAGCCTGGTGGTGTCCTCCTCTGGTCGCCGAGTGGAGTGCTCAGACCAGAAGGCGCCGCCAGCGGGAGAAGACACGCGTCAGTTCGACAAGGCAGT AGCGGTGGTGGCGCAGCAGCTGCTGTCACAGGGCGAGCACTATTGGGAGGTGGAG GTGGGCGACAAACCACGCTGGGCCCTGGGAGTGATGGCGGCTGACGCTTCCCGCC GTGGCCGGCTGCACGCGGTGCCCTCACAGGGGCTGTGGCTGCTGGGTCTGCGCGA TGGCAAGATCCTGGAGGCGCACGTGGAGGCCAAGGAGCCGCGGGCACTGCGCAC CCCAGAGAGGCCTCCGGCGCGCATTGGCCTCTACCTAAGCTTCGCAGATGGCGTCC TGGCTTTCTATGATGCGAGCAACCCCGACGTACTTACGCCAATCTTTTCTTTCCACG AGCGTCTGCCCGGGCCGGTGTACCCCATCTTTGACGTGTGCTGGCACGACAAGGG CAAGAATGCCCAGCCCCTGCTGCTTGTGGGGCCGGAGCAGGAACAGGCCTGA (SEQ ID NO: 59) Example 2

[0292] Vectors were prepared for in vitro delivery of miRNA binding sequences (miR sponges) (FIGs.7A, 7B). When tested in vitro, RFP expression from a CMV promoter in Day 4 differentiated C2C12 myotubes was observed after electroporation with a pU6 plasmid utilized initially to drive ubiquitous expression of the miRNA-Sponge (FIG.8A). RFP expression indicated transfection efficiency as well as reports on specific cells which are expected to express the delivered miRNA-Sponge. eGFP expression under a tMCK promoter after electroporation in Day 5 differentiated C2C12 myotubes showed enhanced expression compared to Day 0 (FIG.8B). eGFP served as a reporter for electroporation efficiency as well as cells that may be expressing the miRNA-Sponge.

[0293] Enhanced MG29 expression in C2C12 myotubes was observed after transfection with miRNA-Sponges by Western Blot of Day 5 differentiated C2C12 cells after transfection of single or combination miRNA-Sponge constructs utilizing pU6. Notably, MG29 protein expression was enhanced in miRNA-Sponge combinations, while only slightly increased with miRNA-Sponge targeting the individual regions (FIG.10).

[0294] Additional vectors were prepared for in vivo delivery. A vector was prepared containing AAV9-miRNA-Sponge expression cassettes for complementary gene transfer, promoting glucose uptake, or reducing plasma membrane damage using a skeletal muscle specific MHCK7 promoter. A construct utilizing a MHCK7 skeletal muscle specific promoter driving eGFP expression served as a reporter for delivery (FIG.9A). A construct utilizing the MHCK7 promoter for driving Glut4 expression was used as a complementary gene therapy delivered to aid in the amount of glucose uptake into skeletal muscle following miRNA- Sponge mediated overexpression of MG29 (FIG.9B). A construct utilizing the MHCK7promoter driving tPA-MG53 expression was used to achieve sustained elevation of circulating MG53 by fusing a tPA secretory peptide at the N-terminus of the MG53 protein. This construct was used as a complementary gene therapy to aid in enhanced damage repair experienced by those with diabetes (FIG.9C).

[0295] MyoAAV-hMG29 and MyoAAV-EGFP vectors were prepared. MyoAAV-hMG29 or MyoAAV-EGFP (1.2 x 1012vg) was delivered intramuscularly into high-fat diet (HFD) mice. After 4 weeks, a [18F] FDG PET scan was performed. Insulin (2U / kg) was injected intraperitoneally (i.p.) immediately before PET imaging. [18F]FDG uptake, expressed as SUVbw, was increased in the MyoAAV-hMG29-injected muscle compared to non-injected muscle or those injected with eGFP as control (FIGs.11A, 11B).

[0296] MyoAAV-hMG29 or MyoAAV-Sponge (1.2 x 1012vg) was delivered intramuscularly into the HFD mice. After 4 weeks, a [18F] FDG PET scan was performed. Insulin (2U / kg) was injected intraperitoneally (i.p.) immediately before PET imaging. [18F]FDG uptake, expressed as SUVbw, was increased in the MyoAAV-Sponge injected muscle (FIG.12B, circles) compared to contralateral control muscle (FIG.12B, squares).

[0297] HFD mice that received MyoAAV-hMG29 or -Sponge (1.2 x 1012vg) intramuscularly showed mitigation of obesity and hyperglycemia, while those receiving eGFP continued to develop obesity and hyperglycemia over the 27 day period (FIG.13).

[0298] C2C12 myoblasts were infected with two different MyoAAV-Sponges and MyoAAV-EGFP, then differentiated for 7 days. Both MyoAAV-Sponges, containing three and four different miRNA binding nucleotide sequences, respectively, increased MG29 expression in the differentiated myotubes (data not shown). The two miRNA-Sponges correspond to SEQ ID NO: 27 and SEQ ID NO: 31. Example 3 Discussion

[0299] A gene named MG29 that is specifically present in skeletal muscle was identified. MG29 facilitates glucose uptake into skeletal muscle. MG29 negative (MG29- / -) mice developed glucose intolerance and insulin-insensitivity, resembling type 2 diabetes (T2D). The MG29 gene contains an unusually long 3′ UTR that inhibited MG29 protein expression. It was found that unwinding of the 3′ UTR could enhance MG29 protein expression, thus improving glucose handling in muscle fibers to mitigate hyperglycemia associated with T2D. In particular, the MG293′ UTR contained binding sites for miRNAs that could be used to boost MG29 expression.

[0300] Intriguingly, mice subjected to a brief training exercise (2 hour run at 12 meter / min) led to acute elevation of MG29 protein expression in skeletal muscle, suggesting a posttranscriptional control mechanism (FIG.3). Interestingly, the unusually long 3′ UTR of MG29 played an important role in regulating MG29 mRNA stability, translation, and / or its tissue specific expression. When the cDNA coding sequence (CDS) for MG29 was transfected into the C2C12 cells, abundant MG29 protein could be detected using western blot. However, transfection of a plasmid containing CDS plus 3′ UTR of MG29 did not result in measurable MG29 protein (FIG.4A). The 3′ UTR-mediated inhibition of MG29 protein expression was also observed in the human MG29 sequence.

[0301] It was demonstrated that MG29 possesses the following unique properties that could be used as druggable targets to treat metabolic diseases e.g., diabetes and / or obesity. First, MG29 protein was predominantly expressed in skeletal muscle and was undetectable in heart, liver, kidney, and brain. Thus, therapeutic interventions of MG29 are unlikely to have off-target effects. Second, MG29’s role in control of TT and Glut4 made increasing MG29 expression uniquely advantageous over other insulin signaling components that control glucose uptake into muscle cells.

[0302] Using a serial deletion approach, a particular domain within the 3′ UTR was identified, which domain when deleted led to enhanced MG29 protein expression (FIG.4A). This was indicative of the MG293′ UTR containing motifs that negatively regulate MG29 protein expression. When the 3′ UTR sequence was transiently expressed in C2C12 cells, a dose dependent recovery of MG29 protein expression was observed (FIG.4B), indicating that the 3′ UTR region sequestered endogenous factors that interacted with the 3′UTR to inhibit MG29 protein expression. Indeed, bioinformatics data mining revealed several motifs within the 3′ UTR of MG29 that could bind to miRNAs, including miR-181A treatment was developed that directly enhanced glucose handling by skeletal muscle and provided a novel approach for treating diabetes. An Adeno-Associated Virus 9 (AAV9) vector was generated, which AAV vector was capable of delivering to a cell the MG29 cDNA, MG293′ UTR or a “miRNA-Sponge,” the latter being a combination of miRNA binding nucleotide sequences that bind to miRNAs, which miRNAs naturally bind to the M29 mRNA 3′UTR and inhibit MG29 expression. By removing such miRNAs the miRNA binding nucleotide sequences provided, e.g., in a nucleic acid, a vector, or an AAV particle, increase MG29 expression in skeletal muscle improving glucose uptake.

[0303] MyoAAV was developed as an AAV vector that has been optimized for gene delivery, specifically targeting muscle tissue (myotropic). MyoAAV vectors encoding a codon-optimized human MG29 or EGFP or EGFP-Sponge sequence under the control of the muscle specific promoter MHCK7 were generated.

[0304] MyoAAV-hMG29 or MyoAAV-eGFP or MyoAAV-Sponge were administrated by intramuscular (gastrocnemius) injection into 25-week-old male mice fed a high-fat diet (HFD, 60 kcal% fat). Each mouse received by i.m. injection a dose of 1.2x1012vg / mouse in a total volume of 180 μl divided into six injection sites distributed in the hind limb.

[0305] To assess the effects of human MG29 or Sponge expression on metabolic activity in the muscle, positron emission tomography (PET) imaging was performed. After 4 weeks, the mice were fasted for 12-15 hours, and [18F]-Fluorodeoxyglucose ([18F]-FDG) was administered intravenously via tail vein injection at a dose of 11-13 MBq. A dynamic PET scan was performed for 60 minutes under a mixture of oxygen (95%) and isoflurane (5% for induction and ≤ 2% for maintenance) anesthesia using an anesthesia machine. Insulin (2 U / kg) was administered intraperitoneally (i.p.) immediately before the PET scan. The PET scans were analyzed to evaluate changes in glucose uptake within the muscle tissue and quantified using the standardized uptake value normalized to body weight (SUVbw). This metric provided an indication of glucose metabolism and was used as a surrogate for assessing muscle activity and overall metabolic function. These measurements were performed in response to the administration of MyoAAV2A-hMG29 or the control Sponge treatment, allowing for a comparative analysis of the metabolic effects induced by each intervention.

[0306] Provided are methods of increasing MG29 expression in skeletal muscle of a subject comprising contacting at least one nucleic acid, that is at least 16 nucleotides in length and has at least 90% sequence identity to a 16-nucleotide sequence of the MG293′ UTR, with the MG293′ UTR of the subject. The nucleic acid is a nucleic acid molecule of any one of SEQ ID NO: 1-59 or 62-65 or the nucleic acidis a nucleic acid molecule having at least 90% homology to any one of SEQ ID NO: 1 to SEQ ID NO: 1-59 or 62-65.

[0307] Provided are methods of improving glucose uptake, regulating glycolysis, or treating type 2 diabetes in a patient comprising administering to the patient at least one nucleic acid, that is at least 16 nucleotides in length and has at least 90% sequence identity to a 16 nucleotide sequence of the M293′ UTR of the patient.

[0308] This disclosure provides a deliverable plasmid containing specific oligonucleotides complementary to miRNA targeting sypl2 (e.g., MG29) mRNA. The plasmid, called 'OliSponge,' utilizes a repeat sequence with a linker to interact with miRNAs. The therapeutic approach involves using AAV9 delivery of the MG29 cDNA or the "miRNA-Sponge," e.g., the “OliSponge” to increase MG29 expression in skeletal muscle and treat diabetes byenhancing glucose uptake in skeletal muscle. The disclosure provides a method of using the 'OliSponge' to counteract negative regulatory miRNAs targeting MG29. Additionally, nanoparticle-guided delivery of the effective miR-sponges can be directly used to boost MG29 expression in skeletal muscle, and in turn mitigate hyperglycemia associated with Type 2 Diabetes (T2D).

[0309] The disclosure provides a method of improving glucose uptake in dysregulated cell types, with a focus on conditions like Type 2 Diabetes Mellitus, or other human diseases that involve dysregulation of glucose homeostasis, where glucose uptake is hindered due to insulin impairment. Delivering the 'OliSponge,' which binds and neutralizes negative regulatory miRNAs of MG29 enhances glucose uptake and treats the associated hyperglycemia in such conditions.

[0310] A therapeutic was provided in which the 3′ UTR of MG29, or a “miRNA-Sponge” was delivered into muscle fibers and enhanced MG29 expression which increased glucose uptake. This therapeutic approach is useful for increasing glucose uptake in diabetic patients, particularly in patients with type-2 diabetes. PET imaging demonstrated that elevated MG29 protein expression can boost glucose uptake into skeletal muscle to mitigate hyperglycemia in diabetes (see FIGs.11 and 12), EQUIVALENTS AND SCOPE

[0311] While several inventive aspects have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive aspects described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive aspects described herein. It is, therefore, to be understood that the foregoing aspects are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive aspects may be practiced otherwise than as specifically described and claimed. Inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two ormore such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0312] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0313] All references, patents, and patent applications described herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0314] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0315] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one aspect, to A only (optionally including elements other than B); in another aspect, to B only (optionally including elements other than A); in yet another aspect, to both A and B (optionally including other elements); etc.

[0316] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0317] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one aspect, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another aspect, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another aspect, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0318] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0319] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that aspects described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative aspects, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative aspects “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

Claims

CLAIMS What is claimed is:

1. A nucleic acid comprising at least two different micro RNA (miRNA) binding nucleotide sequences of a 3′ untranslated region (3′ UTR) of a Mitsugumin 29 (MG29) mRNA.

2. The nucleic acid of claim 1, wherein the at least two different miRNA binding nucleotide sequences are selected from the group consisting of a miRNA-29 from the 3′ arm of a precursor (miR-29a-3p) binding nucleotide sequence or a sequence having at least 90% identity to a miR-29a-3p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA; a miRNA-181a from the 5′ arm of a precursor (miR-181a-5p) binding nucleotide sequence or a sequence having at least 90% identity to a miR- 181a-5p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA; a miRNA- 375 (miR-375-3p) binding nucleotide sequence of the 3′ UTR of the MG29 mRNA or a sequence having at least 90% identity to a miR-375-3p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA; and a miRNA-29a2 from the 3′ arm of a precursor (miR-29a2-3p) binding nucleotide sequence of the 3′ UTR of the MG29 mRNA or a sequence having at least 90% identity to a miR-29a2-3p binding nucleotide sequence of the 3′ UTR of the MG29 mRNA.

3. The nucleic acid of claim 1 or 2, wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO:

1.

4. The nucleic acid of any one of claims 1-3, wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO:

1.

5. The nucleic acid of any one of claims 1-4, wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO: 2.

6. The nucleic acid of any one of claims 1-5, wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO:

2.

7. The nucleic acid of claim 5 or 6, wherein the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 2 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO:

2.

8. The nucleic acid of any one of claims 1-7, wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO:

3.

9. The nucleic acid of any one of claims 1-8, wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO:

3.

10. The nucleic acid of claim 8 or 9, wherein the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 3 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO:

3.

11. The nucleic acid of any one of claims 1-10, wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO:

4.

12. The nucleic acid of any one of claims 1-11, wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO:

4.

13. The nucleic acid of any one of claims 1-12, wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO:

5.

14. The nucleic acid of any one of claims 1-13, wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO: 5.

15. The nucleic acid of claim 13 or 14, wherein the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 5 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO:

5.

16. The nucleic acid of any one of claims 1-15, wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO:

6.

17. The nucleic acid of any one of claims 1-16, wherein at least one of the at least two miRNA binding nucleotide sequences comprises a sequence of SEQ ID NO:

6.

18. The nucleic acid of claim 16 or 17, wherein the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 6 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO:

6.

19. The nucleic acid of any one of claims 1-18, wherein at least one of the at least two miRNA binding nucleotide sequences is at least 90% identical to a nucleotide sequence of SEQ ID NO:

7.

20. The nucleic acid of any one of claims 1-19, wherein at least one of the at least two miRNA binding nucleotide sequences comprise a sequence of SEQ ID NO:

7.

21. The nucleic acid of claim 19 or 20, wherein the miRNA binding nucleotide sequence at least 90% identical to or comprising SEQ ID NO: 7 does not extend more than 10 nucleotides beyond the 3’ or 5’ end of SEQ ID NO:

7.

22. The nucleic acid of any one of claims 1-21, wherein the nucleic acid further comprises a leader sequence.

23. The nucleic acid of any one of claims 1-22, wherein the nucleic acid further comprises a linker sequence.

24. The nucleic acid of claim 23, wherein the linker sequence comprises between about 4 nucleotides and about 20 nucleotides.

25. The nucleic acid of claim 22 or 23, wherein the linker sequence comprises between about 4 nucleotides and about 10 nucleotides.

26. The nucleic acid of any one of claims 1-25, wherein the nucleic acid comprises more than one copy of at least one miRNA binding nucleotide sequence.

27. The nucleic acid of claim 26, wherein the nucleic acid comprises between about 3 copies and about 100 copies of at least one miRNA binding nucleotide sequence.

28. The nucleic acid of any one of claims 1-27, wherein the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

2.

29. The nucleic acid of claim 28 wherein the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 25 or 26.

30. The nucleic acid of any one of claims 1-29, wherein the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

3.

31. The nucleic acid of claim 30, wherein the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 17 or 18.

32. The nucleic acid of any one of claims 1-31, wherein the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

33. The nucleic acid of claim 32, wherein the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 21 or 22.

34. The nucleic acid of any one of claims 1-33, wherein the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2 and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

3.

35. The nucleic acid of claim 34, wherein the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 23 or 24.

36. The nucleic acid of any one of claims 1-35, wherein the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2 and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

37. The nucleic acid of claim 36, wherein the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 63 or 64.

38. The nucleic acid of any one of claims 1-37, wherein the at least two different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3 and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

39. The nucleic acid of claim 38, wherein the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 19 or 20.

40. The nucleic acid of any one of claims 1-39, wherein the nucleic acid comprises at least three different miRNA binding nucleotide sequences.

41. The nucleic acid of claim 40, wherein the at least three different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, and a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3.

42. The nucleic acid of claim 41, wherein the at least two miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 27 or 28.

43. The nucleic acid of claim 42, wherein the at least three different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

44. The nucleic acid of claim 43, wherein the at least three miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 29 or 30.

45. The nucleic acid of claim 44, wherein the at least three different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

46. The nucleic acid of claim 45, wherein the at least three miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 65 or 66.

47. The nucleic acid of any one of claims 1-46, wherein the nucleic acid comprises four different miRNA binding nucleotide sequences.

48. The nucleic acid of claim 47, wherein the four different miRNA binding nucleotide sequences comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, and a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

49. The nucleic acid of claim 48, wherein the four miRNA binding nucleotide sequences comprise a nucleotide sequence of SEQ ID NO: 31 or 32.

50. A nucleic acid comprising at least one miRNA binding nucleotide sequence of a 3′ UTR of a MG29 mRNA and an inverted terminal repeat.

51. A nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-59 or 62-65.

52. A vector comprising a nucleic acid of any one of claims 1-51.

53. A viral particle comprising a nucleic acid of any one of claims 1-51 or a vector of claim 52.

54. The viral particle of claim 53, wherein the viral particle is an adeno-associated virus (AAV) particle.

55. The viral particle of claim 54, comprising a capsid protein of an AAV serotype 6, 8, 9, rh74, or MyoAAV, optionally the AAV particle comprises a capsid protein of an AAV serotype MyoAAV.

56. A composition comprising a nucleic acid of any one of claims 1-51, a vector of claim 52, or a viral particle of any one of claims 53-55.

57. The composition of claim 56, further comprising an excipient.

58. The composition of claim 57, wherein the excipient is a pharmaceutically acceptable excipient.

59. A method of increasing Mitsugumin 29 (MG29) expression in a cell, the method comprising contacting the cell with an effective amount of: a nucleic acid of any one of claims 1-51, a vector of claim 52, a composition of any one of claims 56-58, or a viral particle of any one of claims 53-55.

60. A method of increasing glucose uptake in a cell of a subject in need thereof, the method comprising contacting the skeletal muscle of the subject with an effective amount of: a nucleic acid of any one of claims 1-51, a vector of claim 52, a composition of any one of claims 56-58, or a viral particle of any one of claims 53-55.

61. The method of any one of claims 59-60, wherein the cell is in vitro.

62. A method of increasing insulin sensitivity in a subject in need thereof, the method comprising administering to the subject an effective amount of: a nucleic acid of any one of claims 1-51, a vector of claim 52, a composition of any one of claims 56-58, or a viral particle of any one of claims 53-55.

63. A method of reducing blood glucose in a subject in need thereof, the method comprising administering to the subject an effective amount of: a nucleic acid of any one of claims 1-51, a vector of claim 52, a composition of any one of claims 56-58, or a viral particle of any one of claims 53-55.

64. A method of treating a metabolic disease in a subject in need thereof, the method comprising administering to the subject with an effective amount of: a nucleic acid of any one of claims 1-51, a vector of claim 52, or a composition of any one of claims 56-58, or a viral particle of any one of claims 53-55.

65. The method of claim 64, wherein the metabolic disease involves dysregulation of glucose homeostasis.

66. The method of claim 64 or 65, wherein the metabolic disease is hyperglycemia.

67. The method of any one of claims 64-66, wherein the metabolic disease is diabetes.

68. The method of any one of claims 64-67, wherein the metabolic disease is type II diabetes.

69. The method of any one of claims 59-68, wherein the subject is a human.

70. A method of increasing MG29 expression in skeletal muscle of a subject comprising contacting at least one nucleic acid, that is at least 16 nucleotides in length and has at least 90% sequence identity to a 16 nucleotide sequence of the MG293′UTR, with the MG29 3′ UTR of the subject.

71. The method of claim 70, wherein the nucleic acid is of any one of SEQ ID NO: 1-59 or 62-65 or having at least 90% homology to any one of SEQ ID NO: 1-59 or 62-65.

72. A method of improving glucose uptake, regulating glycolysis, or treating type 2 diabetes in a patient comprising administering to the patient at least one nucleic acid, that is at least 16 nucleotides in length and has at least 90% sequence identity to a 16 nucleotide sequence of the M293′UTR, with the MG293′ UTR of the patient.

73. The method of any one of claims 60-72, wherein the step of administering or contacting comprises intramuscular injection.

74. A nucleic acid of any one of claims 1-51, a vector of claim 52, a composition of any one of claims 56-58, or a viral particle of any one of claims 53-55 for use in the treatment of a metabolic disease.

75. A kit comprising a nucleic acid of any one of claims 1-51, a vector of claim 52, a composition of any one of claims 56-58, or a viral particle of any one of claims 53-55.

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