Methods and compositions of short small hairpin rnas and micrornas for wound healing

Abstract

Wound healing is a complex homeostatic process in which several distinct types coordinate to repair a physical damage. Failure to close wounds contributes to the pathology of conditions like diabetes mellitus, particularly in the elderly. Presented herein are molecules, pharmaceutical compositions, and methods for applying small RNA oligonucleotide technology to wound healing. Small RNA oligonucleotide approaches as disclosed herein provide a therapeutic strategy for improving both basal and pathological wound healing.

What is claimed is: 
     
         1 . A short small hairpin RNA (sshRNA) for inhibiting prolyl hydroxylase domain-containing protein 2 (PHD2) comprising:
 (a) an antisense sequence that is capable of hybridizing to an RNA transcript encoding prolyl hydroxylase domain-containing protein 2; and   (b) a sense sequence,
 wherein the sense and antisense sequences form a stem having a length of less than or equal to 19 base pairs. 
   
     
     
         2 . The sshRNA of  claim 1 , wherein the RNA transcript is encoded by an Eg1 nine homolog 1 (EGLN1) gene, wherein the EGLN1 gene, minus introns, is represented by a sequence selected from SEQ ID NO. 1 and SEQ ID NO. 2. 
     
     
         3 . The sshRNA of  claim 1 , comprising a loop region selected from a direct connection, 1 nucleotide and 2 nucleotides in length. 
     
     
         4 . The sshRNA of  claim 1 , wherein the sshRNA consists of the antisense sequence, the sense sequence, a loop region, an optional overhang sequence, and an optional conjugate moiety. 
     
     
         5 . The sshRNA of  claim 4 , wherein the loop region has a length of zero nucleotides, 1 nucleotide or 2 nucleotides. 
     
     
         6 . The sshRNA of  claim 5 , wherein the loop region consists of zero nucleotides, 1 nucleotide or 2 nucleotides. 
     
     
         7 . The sshRNA of  claim 1 , wherein the sshRNA consists of the antisense sequence, the sense sequence, an optional overhang sequence, and an optional conjugate moiety, wherein the sense sequence and the antisense sequence are directly connected. 
     
     
         8 . The sshRNA of  claim 1 , wherein the antisense sequence is from about 80% complementary to 100% complementary to the PHD2 transcript. 
     
     
         9 . The sshRNA of  claim 1 , wherein the sense sequence is from about 10 nucleotides to about 19 nucleotides in length. 
     
     
         10 . The sshRNA of  claim 1 , wherein the sense sequence is from about 11 nucleotides to about 19 nucleotides in length. 
     
     
         11 . The sshRNA of  claim 1 , wherein at least one nucleotide comprises a chemical modification. 
     
     
         12 . The sshRNA of  claim 11 , wherein the chemical modification increases the stability of the shRNA in a biological fluid at least about 20%. 
     
     
         13 . The sshRNA of  claim 1 , wherein the sshRNA is represented by a sequence selected from SEQ ID NOS: 5-8, 11-31, 49-325. 
     
     
         14 . A microRNA (miRNA) antagonist comprising an antisense strand capable of hybridizing to and inhibiting miR-210. 
     
     
         15 . The miRNA antagonist of  claim 14 , wherein the miR-210 is represented by SEQ ID NO. 3. 
     
     
         16 . The miRNA antagonist of  claim 14 , wherein the miRNA antagonist reduces an amount or an activity of the miR-210 from about 10% to about 99.9%. 
     
     
         17 . The miRNA antagonist of  claim 14 , wherein at least one ribonucleotide comprises a chemical modification. 
     
     
         18 . The miRNA antagonist of  claim 14 , wherein every ribonucleotide comprises a chemical modification. 
     
     
         19 . The miRNA antagonist of  claim 17  or  18 , wherein the chemical modification is a sugar modification. 
     
     
         20 . The miRNA antagonist of  claim 19 , wherein the sugar modification comprises a 2′-O-methyl modification, a LNA modification, a DNA modification, or a 2′-F modification. 
     
     
         21 . The miRNA antagonist of  claim 19 , wherein the sugar modification is a 2′-O-methyl modification. 
     
     
         22 . The miRNA antagonist of  claim 19 , wherein the sugar modification is a LNA modification. 
     
     
         23 . The miRNA antagonist of  claim 14 , wherein the miRNA antagonist comprises phosphorothioate internucleotide linkages. 
     
     
         24 . The miRNA antagonist of  claim 14 , wherein the miRNA antagonist comprises a backbone modification. 
     
     
         25 . The miRNA antagonist of  claim 24 , wherein the backbone modification is selected from a C3 spacer or ZEN. 
     
     
         26 . The miRNA antagonist of  claim 14 , wherein the miRNA antagonist comprises a 2′-O-methyl modification at every position and three phosphorothioate internucleotide linkages at consecutive residues at both the 5′- and 3′-end. 
     
     
         27 . The miRNA antagonist of  claim 14 , wherein the miRNA antagonist comprises phosphorothioate linkages at every position and 2′-O-methyl modifications at positions 1, 3, 4, 6, 7, 9, 10, 12, 13, and 15 and LNA modifications at positions 2, 5, 8, 11, and 14. 
     
     
         28 . The miRNA antagonist of  claim 14 , wherein the miRNA antagonist comprises 2′-O-methyl modifications at every position and ZEN modifications between position 1 and 2 and also at the 3′-end. 
     
     
         29 . The miRNA antagonist of  claim 14 , wherein the miRNA antagonist is represented by a sequence selected from SEQ ID NOS: 32-43, 326-328. 
     
     
         30 . A pre-miRNA mimic for increasing a steady state level of a mature miR-21 comprising:
 (a) a sense sequence;   (b) an antisense sequence; and   (c) a loop region, wherein the length of the loop region is the length of 2 nucleotides or less.   
     
     
         31 . The pre-miRNA of  claim 30 , wherein the mature miR-21 is represented by SEQ ID NO.4. 
     
     
         32 . The pre-miRNA mimic of  claim 30 , wherein the loop region consists of a direct connection, or consists of 1-2 nucleotides or nucleotide moieties. 
     
     
         33 . The pre-miRNA mimic of  claim 30 , wherein the pre-miRNA mimic is represented by a sequence selected from SEQ ID NOS: 44-48. 
     
     
         34 . A pharmaceutical composition comprising a pharmaceutically-acceptable substrate, carrier or salt, and at least one oligonucleotide selected from:
 (a) the sshRNA of  claim 1 ;   (b) the miRNA antagonist of  claim 14 ; and   (c) the pre-miRNA mimic of  claim 30 .   
     
     
         35 . A pharmaceutical composition comprising a pharmaceutically-acceptable substrate, carrier or salt, and a combination of oligonucleotides selected from:
 (a) the sshRNA of  claim 1 ;   (b) the miRNA antagonist of  claim 14 ; and   (c) the pre-miRNA mimic of  claim 30 .   
     
     
         36 . The pharmaceutical composition of  claim 34  or  35 , wherein the pharmaceutically-acceptable substrate is a mesh or a dressing. 
     
     
         37 . The pharmaceutical composition of  claim 36 , wherein the mesh is a layered mesh. 
     
     
         38 . The pharmaceutical composition of  claim 34  or  35 , wherein the composition is formulated for topical administration. 
     
     
         39 . A method for treating a wound in a subject in need thereof comprising administering a therapeutically effective amount of a composition selected from:
 (a) the sshRNA of  claim 1 ;   (b) the miRNA antagonist of  claim 14 ;   (c) the pre-miRNA mimic of  claim 30 ; and   a combination thereof.   
     
     
         40 . The method of  claim 39 , wherein the wound is a chronic wound. 
     
     
         41 . The method of  claim 39 , wherein the wound is a non-healing wound. 
     
     
         42 . The method of  claim 39 , wherein the subject has diabetes mellitus. 
     
     
         43 . The method of  claim 39 , wherein the wound is a skin wound. 
     
     
         44 . The method of  claim 39 , comprising administering the composition topically. 
     
     
         45 . The method of  claim 39 , comprising a dressing, wherein the dressing comprises the composition. 
     
     
         46 . An oligonucleotide according to any one of  claim 1 ,  14 , or  30 , and any combination thereof, for use in a method of treating a wound in an animal or human. 
     
     
         47 . An R oligonucleotide NA according to any one of  claim 1 ,  14 , or  30 , and any combination thereof, for treatment of a wound in a subject. 
     
     
         48 . A kit comprising:
 (a) the sshRNA of  claim 1 ;   (b) the miRNA antagonist of  claim 14 ; and   (c) the pre-miRNA mimic of  claim 30 .