# Patent

## Title
Fidgetin-like 2 as a target to enhance wound healing

## Abstract

Methods of treating a wound in a subject are provided comprising administering to the subject an amount of an inhibitor of Fidgetin-like 2. Compositions and pharmaceutical compositions comprising an amount of an inhibitor of Fidgetin-like 2 are also provided. Methods are also provided for identifying an inhibitor of Fidgetin-like 2.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/553,155, filed Jul. 19, 2012, now allowed, which claims the benefit of U.S. Provisional Patent Application No. 61/510,172, filed Jul. 21, 2011, the contents of each of which are herein incorporated by reference in their entirety.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under grant number GM065940 awarded by the National Institute of General Medical Sciences. The government has certain rights in the invention.


SEQUENCE LISTING INCOPORATION
The “.txt” Sequence Listing filed with this application by EFS and which is entitled 96700—1855_ST25.txt, is 26 kilobytes in size and which was created on Jun. 11, 2012, is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The development of safe and effective therapies for treating acute and chronic wounds is an issue currently of great interest to clinical scientists and industry, alike. Wound healing is an intricate, multi-stage process that relies heavily on the delivery of new cells to the wound zone. Two key elements of the wound healing response are fibroplasia and epithelialization when fibroblasts and epithelial cells, respectively, enter the wound to form a protective barrier from the external environment. This is stimulated by cell proliferation and migration from the wound edge. The identification of agents that increase the rate at which cells invade and close a wound would represent a major advance in wound healing therapeutics. Ideally, this would be a topically applied agent that stimulates the proliferation and migration of fibroblasts and wound edge epithelial cells.
The present invention addresses this need and identifies a novel target in promoting wound healing and provides therapies and assays based thereon.
SUMMARY OF THE INVENTION
A method of treating a wound in a subject is provided comprising administering to the subject an amount of an inhibitor of Fidgetin-like 2 effective to treat the wound.
A pharmaceutical composition is provided comprising an amount of an inhibitor of Fidgetin-like 2.
A method for identifying a candidate agent for treating a wound comprising:
a) determining the activity of an amount of Fidgetin-like 2; and
b) contacting the amount of Fidgetin-like 2 with the candidate agent and determining the activity of the amount of Fidgetin-like 2 in the presence of the candidate agent, wherein a decreased activity of the amount of Fidgetin-like 2 in the presence of the candidate agent as compared to the activity of Fidgetin-like 2 in the absence of the candidate agent indicates that the candidate agent can treat a wound, and wherein no change in or an increased activity of the amount of Fidgetin-like 2 in the presence of the candidate agent as compared to the activity of Fidgetin-like 2 in the absence of the candidate agent does not indicate that the candidate agent can treat a wound.
An inhibitor of Fidgetin-like 2 is provided for treating a wound or promoting wound healing.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-1B: Fidgetin-like 2 is expressed in human tissue culture cells where it localizes to microtubules. FIG. 1A shows a Western blot of human U2OS cell lysates probed with an anti-Fidgetin-like 2 antibody generated in-lab. This antibody recognizes a single band that is substantially decreased by Fidgetin-like 2 siRNA treatment (see FIG. 2 below). FIG. 1B shows a migrating U2OS cell double-labeled for Fidgetin-like 2 and microtubules. At high magnification (inset), Fidgetin-like 2 clearly co-localizes with spans of the microtubule lattice near the cell edge.
FIG. 2A-2E: Cells depleted of Fidgetin-like 2 display a several-fold increase in their rate of wound healing and migration. FIG. 2A shows Western blots of U2OS cell lysates obtained from control (N) and Fidgetin-like 2 siRNA treated cultures (72 hrs. after treatment). Actin was used as a loading control. FIG. 2B shows time-lapse phase contrast images of “wound healing” assays performed in control and Fidgetin-like 2 siRNA treated cultures. In these assays, a monolayer of U2OS cells is “wounded” by a pipette tip and the invasion of cells into the wound is monitored over time. FIG. 2C shows the average rate of wound closure in each condition which is increased nearly 4-fold after Fidgetin-like 2 siRNA. FIG. 2D shows the trajectories of single control and Fidgetin-like 2 siRNA treated cells as they enter the wound zone. Not only do Fidgetin-like 2 siRNA-treated cells move several-fold faster than controls, they also display more directionally persistent migration as indicated in FIG. 2E.
FIG. 3: Fidgetin-like 2 siRNA also dramatically enhances chemotaxis of cultured human cells. The impact of Fidgetin-like 2 siRNA on chemotaxis of human U2OS cells was measured in a transwell assay (modified Boyden chamber). This assay counts the number of cells that move through 8 μm pores towards a chemoattractant loaded in the distal well. The graph in FIG. 3 shows the number of control (N) and siRNA-treated (F12) cells that migrated through the pores before and three hours after the addition of a chemoattractant (Fetal Bovine Serum).
FIG. 4: siRNA directed to Fidgetin-like 2 elevates the rate of U2OS cell proliferation.
FIG. 5: Kymographs showing (left) severing of a MT incubated 50 nM recombinant Fidgetin and (right) depolymerization at the minus-end of a polarity marked induced MT incubated with 25 nM Fidgetin. ATP was added in both conditions. The reaction is entirely inhibited by the non-hydrolyzable ATP analogue, AMPPNP.
FIG. 6: Left panels show time-lapse phase contrast images of “wound healing” assays performed in control and Fidgetin-like 2 siRNA treated cultures of human dermal fibroblasts. In these assays, a monolayer is “wounded” by a pipette tip and the invasion of cells into the wound is monitored over time. The right panel shows the average rate of wound closure measured in each condition.
FIG. 7A-7B: Topical application of Fidgetin-like 2 siRNA encapsulated in nanpoparticles (np-si) increases the rate of wound closure in vivo. A) Images showing the closure of control and Fidgetin-like 2 np-si treated full thickness biopsy wounds positioned next to one another on the flank of a mouse. B) Plot showing the average rate of wound closure from each condition (n=3). Error bars are SEM. Wound closure is plotted to 50% because smaller wounds are difficult to measure with accuracy.
FIG. 8: Histology of Control and Fidgetin-like 2 np-si treated mouse biopsy wounds: Hematoxylin and Eosin staining of 5 mm punch biopsy wounds, mouse skin, day 9 after wounding. Control wounds (left) demonstrate a thin layer of re-epitheliazed epidermis with mounds of serum crust overlying. The dermis is composed of inflammatory, disorganized granulation tissue without evidence of subcutis. Fidgetin-like 2 np-si treated wounds demonstrate a completely re-epithelialized epidermis with overlying organized basket weaving stratum corneum. The dermis is devoid of intense inflammation and is infiltrated with parallel fibroblasts. There is a healthy appearing subcutis present.
FIG. 9: Trichome masson staining for collagen reveal a necrobiotic degenerating collagen with minimal pale, newly deposited collagen present in the wound bed in the control (left) wound. In the Fidgetin-like 2 np-si treated wound, minimal cell death (as indicated by red) is noted and homogenous newly formed collagen (light blue) is noted.
FIG. 10: Neoangiogenesis in embryonic hearts treated with Control or Fidgetin-like 2 np-si. Images show representative control and Fidgetin-like 2 np-si treated hearts three days after np-si treatment. In the control, migrating endocaridal cells (GFP-labeled) have penetrated the ventricular wall (arrows) and will undergo angiogenesis over the next several days. By contrast, Fidgetin-like 2 np-si treatment dramatically promotes the angiogenic process by the endocardial cells, which have already formed a fine vascular network at this time point (arrows).
FIG. 11: Fidgetin and Fidgetin-like 2 siRNA nanoparticles strongly promote axon regrowth from hippocampal neurons. Images show dissociated rat hippocampal neurons 48 hours after siRNA treatment. (A similar effect was observed after 24 hours; not shown).
FIG. 12: Graphical representation showing Fidgetin and Fidgetin-like 2 siRNA nanoparticles strongly promote axon regrowth from hippocampal neurons.
FIG. 13: Fidgetin and Fidgetin-like 2 siRNA nanoparticles promote axon growth and differentiation in N2A neuroblastoma cells (cells stained for microtubules).



DETAILED DESCRIPTION OF THE INVENTION
A method of treating a wound in a subject is provided comprising administering to the subject an amount of an inhibitor of Fidgetin-like 2 effective to treat a wound.
In an embodiment, the amount of inhibitor of Fidgetin-like 2 is effective to accelerate wound healing.
In an embodiment, the inhibitor of Fidgetin-like 2 is administered locally to the wound. In an embodiment, the inhibitor of Fidgetin-like 2 is administered via a vein or artery. In an embodiment, the inhibitor of Fidgetin-like 2 is administered by injection, catheterization or cannulation. In an embodiment, the inhibitor of Fidgetin-like 2 is administered from an implant that elutes the inhibitor, for example a eluting stent or an eluting skin patch.
In an embodiment, the inhibitor of Fidgetin-like 2 is administered topically to the wound.
In an embodiment, the inhibitor of Fidgetin-like 2 is a nucleic acid. In an embodiment, the inhibitor of Fidgetin-like 2 is an siRNA or shRNA. In an embodiment, the nucleic acid is directed against a DNA encoding Fidgetin-like 2 or against an mRNA encoding Fidgetin-like 2.
In an embodiment of the method, the inhibitor of Fidgetin-like 2 is encapsulated in a nanoparticle. In an embodiment the nanoparticle is a liposomal nanoparticle.
In an embodiment, the Fidgetin-like 2 is human Fidgetin-like 2.
In an embodiment, the Fidgetin-like 2 comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:2.
In an embodiment, the wound is an epidermal wound. In an embodiment, the wound is a skin wound.
In an embodiment, the wound is a cardiac tissue wound. In an embodiment, the wound is a cardiovascular wound, for example resulting from a myocardial infarction.
In an embodiment, the wound is a neuronal wound.
A pharmaceutical composition is provided comprising an amount of an inhibitor of Fidgetin-like 2. In an embodiment, the pharmaceutical composition comprises an amount of an inhibitor of Fidgetin-like 2 effective to treat a wound in a human subject. In an embodiment, the wound is a skin wound. In an embodiment, the wound is an epidermal wound.
In an embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
In an embodiment of the pharmaceutical composition the inhibitor of Fidgetin-like 2 is a nucleic acid.
In an embodiment of the pharmaceutical composition the inhibitor of Fidgetin-like 2 is an siRNA or shRNA.
In an embodiment of the pharmaceutical composition the nucleic acid is directed against a DNA encoding Fidgetin-like 2 or against an mRNA encoding Fidgetin-like 2.
In an embodiment of the pharmaceutical composition, the inhibitor of Fidgetin-like 2 is encapsulated in a nanoparticle. In an embodiment the nanoparticle is a liposomal nanoparticle.
In an embodiment of the pharmaceutical composition the Fidgetin-like 2 is human Fidgetin-like 2.
In an embodiment of the pharmaceutical composition the Fidgetin-like 2 comprises SEQ ID NO:2.
A method for identifying a candidate agent for treating a wound comprising:
a) determining the activity of an amount of Fidgetin-like 2; and
b) contacting the amount of Fidgetin-like 2 with the candidate agent and determining the activity of the amount of Fidgetin-like 2 in the presence of the candidate agent, wherein a decreased activity of the amount of Fidgetin-like 2 in the presence of the candidate agent as compared to the activity of Fidgetin-like 2 in the absence of the candidate agent indicates that the candidate agent can treat a wound, and wherein no change in or an increased activity of the amount of Fidgetin-like 2 in the presence of the candidate agent as compared to the activity of Fidgetin-like 2 in the absence of the candidate agent does not indicate that the candidate agent can treat a wound.
In an embodiment, the Fidgetin-like 2 is human Fidgetin-like 2.
In an embodiment, the Fidgetin-like 2 comprises SEQ ID NO:2.
In an embodiment, the candidate agent is a small molecule of 2000 Daltons or less. In an embodiment, the candidate agent is a small molecule of 1000 Daltons or less. In an embodiment, the candidate agent is a substituted or un-substituted hydrocarbon small molecule.
An inhibitor of Fidgetin-like 2 is provided for treating a wound or promoting wound healing.
In an embodiment, the inhibitor of Fidgetin-like 2 is a nucleic acid.
In an embodiment, the inhibitor is an siRNA or shRNA.
In an embodiment, the nucleic acid is directed against a DNA encoding Fidgetin-like 2 or against an mRNA encoding Fidgetin-like 2.
In an embodiment, the Fidgetin-like 2 is human Fidgetin-like 2.
In an embodiment, the Fidgetin-like 2 comprises SEQ ID NO:2.
In an embodiment, the inhibitor or the candidate agent is an aptamer, a nucleic acid, an oligonucleotide, or a small organic molecule of 2000 Daltons or less. In an embodiment, the inhibitor is cell-membrane permeable.
The dosage of the inhibitor administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific inhibitor and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with the inhibitor and the desired therapeutic effect.
A dosage unit of the inhibitor may comprise a single compound, or a mixture of the compound with one or more anti-infection compound(s) or wound healing-promoting compound(s).
In an embodiment, the siRNA (small interfering RNA) as used in the methods or compositions described herein comprises a portion which is complementary to an mRNA sequence encoding a Fidgetin-like 2 protein. In an embodiment, the Fidgetin-like 2 protein is a human Fidgetin-like 2 protein. In an embodiment, the mRNA is encoded by the DNA sequence NCBI Reference Sequence: NM—001013690.4 (SEQ ID NO:1), and the siRNA is effective to inhibit expression of Fidgetin-like 2 protein. In an embodiment, the Fidgetin-like 2 protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:2.
In an embodiment, the siRNA comprises a double-stranded portion (duplex). In an embodiment, the siRNA is 20-25 nucleotides in length. In an embodiment the siRNA comprises a 19-21 core RNA duplex with a one or two nucleotide 3′ overhang on, independently, either one or both strands. The siRNA can be 5′ phosphorylated, or not, and may be modified with any of the known modifications in the art to improve efficacy and/or resistance to nuclease degradation. In an embodiment the siRNA can be administered such that it is transfected into one or more cells. In an embodiment, the siRNA is 5′ phosphorylated.
In an embodiment, the 5′ terminal residue of a strand of the siRNA is phosphorylated. In an embodiment the 5′ terminal residue of the antisense strand of the siRNA is phosphorylated. In one embodiment, a siRNA of the invention comprises a double-stranded RNA wherein one strand of the double-stranded RNA is 80, 85, 90, 95 or 100% complementary to a portion of an RNA transcript of a gene encoding Fidgetin-like 2 protein. In an embodiment, the RNA transcript of a gene encoding Fidgetin-like 2 protein is an mRNA. In an embodiment, the Fidgetin-like 2 protein is a human Fidgetin-like 2 protein. In an embodiment, a siRNA of the invention comprises a double-stranded RNA wherein one strand of the RNA comprises a portion having a sequence the same as a portion of 18-25 consecutive nucleotides of an RNA transcript of a gene encoding Fidgetin-like 2 protein. In an embodiment, the Fidgetin-like 2 protein is a human Fidgetin-like 2 protein. In yet another embodiment, a siRNA of the invention comprises a double-stranded RNA wherein both strands of RNA are connected by a non-nucleotide linker. Alternately, a siRNA of the invention comprises a double-stranded RNA wherein both strands of RNA are connected by a nucleotide linker, such as a loop or stem loop structure.
In one embodiment, a single strand component of a siRNA of the invention is from 14 to 50 nucleotides in length. In another embodiment, a single strand component of a siRNA of the invention is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 21 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 22 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 23 nucleotides in length. In one embodiment, a siRNA of the invention is from 28 to 56 nucleotides in length. In another embodiment, a siRNA of the invention is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 nucleotides in length.
In another embodiment, an siRNA of the invention comprises at least one 2′-sugar modification. In another embodiment, an siRNA of the invention comprises at least one nucleic acid base modification. In another embodiment, an siRNA of the invention comprises at least one phosphate backbone modification. As used herein, “at least one” means one or more.
In one embodiment, RNAi inhibition of Fidgetin-like 2 protein is effected by a short hairpin RNA (“shRNA”). The shRNA is introduced into the appropriate cell by transduction with a vector. In an embodiment, the vector is a lentiviral vector. In an embodiment, the vector comprises a promoter. In an embodiment, the promoter is a U6 or H1 promoter. In an embodiment the shRNA encoded by the vector is a first nucleotide sequence ranging from 19-29 nucleotides complementary to the target gene/mRNA, in the present case the mRNA encodes Fidgetin-like 2 protein. In an embodiment the Fidgetin-like 2 protein is a human Fidgetin-like 2 protein. In an embodiment the shRNA encoded by the vector also comprises a short spacer of 4-15 nucleotides (a loop, which does not hybridize) and a 19-29 nucleotide sequence that is a reverse complement of the first nucleotide sequence. In an embodiment the siRNA resulting from intracellular processing of the shRNA has overhangs of 1 or 2 nucleotides. In an embodiment the siRNA resulting from intracellular processing of the shRNA overhangs has two 3′ overhangs. In an embodiment the overhangs are UU.








NCBI Reference Sequence: NM_001013690.4



(nucleic acid encoding Human Fidgetin-like 2)


(SEQ ID NO: 1)










   1
agtgagctat ggggacacta ctgcactgta gcctgggcaa cagagcaaga ccttgtctca



 


  61
aaaatgtata tatattttgg gctttttttc ctaaaacggg aactacaaca gcatatttgc


 


 121
gagctgatga gagtgaccca gcagagaggg aaatggatca gctctgttga agatgcactg


 


 181
gacaccagaa cacgcccagc ccctcaacca gtggccagag cagcacctgg acgtctcctc


 


 241
caccaccccg tcgccggccc acaagttgga gttgccccct gggggtcgcc aacgctgcca


 


 301
ctacgcttgg gcacacgacg acatctcagc cctcactgcc tccaacctcc taaagcgcta


 


 361
tgcagagaag tactctgggg tcttggattc tccctacgag cgtccggccc tgggcgggta


 


 421
cagcgacgcc tccttcctca acggcgccaa aggggatccc gagccctggc cagggccgga


 


 481
gccaccctac cccttggcct cactccacga aggcctccca ggaaccaaat cgggcggtgg


 


 541
cggcggttcc ggggccctgg ggggctcccc agttttagcc gggaacctcc ctgaacccct


 


 601
ctacgccggc aatgcgtgcg ggggcccatc ggcggcgccc gagtacgcgg ccggctacgg


 


 661
cggggggtac ctggcgccgg gttactgcgc gcagacgggc gccgcgctgc ccccgccgcc


 


 721
cccggccgcg ctcctgcagc ccccaccgcc tccggggtac gggccctcag cgccgctgta


 


 781
caactatccc gcagggggct acgcagcgca gcccggctat ggcgcgctcc cgccgccccc


 


 841
aggcccaccc ccggccccct acctgacccc gggcctgccc gcgcccacgc ccctgcccgc


 


 901
gccggcaccg cccaccgcct atggcttccc cacggccgcg ccgggtgccg aatccgggct


 


 961
gtcgctgaag cgcaaggccg ccgacgaggg gcccgagggc cgctaccgca agtacgcgta


 


1021
cgagcccgcc aaggcccccg tggctgacgg agcctcctac cccgccgcgg acaacggcga


 


1081
atgtcggggc aacgggttcc gggccaagcc gccaggagcc gcggaggagg cgtcgggcaa


 


1141
gtacggtggc ggcgtccccc tcaaggtcct gggctccccc gtctacggcc cgcaactgga


 


1201
gccctttgaa aagttcccgg agcgggcccc ggctcctcgt ggggggttcg ccgtgccgtc


 


1261
gggggagact cccaaaggcg tggaccctgg ggccctggag ctggtgacga gcaagatggt


 


1321
ggactgcggg cccccggtgc agtgggcgga tgtggcgggc cagggcgcgc tcaaggcggc


 


1381
gctggaggag gagctggtgt ggcccctgct caggccgccc gcctacccgg gcagcctgcg


 


1441
cccgccgcgg accgtcctgc tctttgggcc gcggggcgcg ggcaaagcgc tgctgggccg


 


1501
ctgcctcgcc acgcagctgg gcgccacgct gttgcgcctg cgcggcgcga ccctggctgc


 


1561
gcccggcgcc gccgagggcg cgcgcctcct ccaggccgcc ttcgcggccg cgcgctgccg


 


1621
cccaccctcc gtactcctca tcagcgagct agaggcgctg ctccccgccc gggacgacgg


 


1681
cgcggcggca gggggcgcgc tgcaggtgcc gctcctggcc tgcctggacg ggggctgcgg


 


1741
cgcgggggct gacggcgtgc tggttgtggg caccacctcg cggcccgcgg ctctggacga


 


1801
ggcgacccgc cggcgcttct ctctccgctt ctacgtggcg ctgcccgaca gcccggcccg


 


1861
cgggcagatc ctgcagcggg cgctggccca gcagggctgc gcgctcagtg agcgggaact


 


1921
ggcggcgctg gtgcagggca cgcagggctt ctctgggggc gagctggggc agctgtgcca


 


1981
gcaggcggcg gccggggcgg gcctcccggg gctgcagcgc cccctctcct acaaggacct


 


2041
ggaggcggcg ctggccaagg tgggccctag ggcctctgcc aaggaactgg actcgttcgt


 


2101
ggagtgggac aaaatgtacg gctccggaca ctgacggcgc gcgggggagg ccgcgggagc


 


2161
cgcagtccct ccgtccccgc cgcctccgcg tgggagggat gtcactgact aaacccggct


 


2221
ggcaggggct ggagtggtga atgtgggatc ggggacagga ggggtctgcc ggtggatatt


 


2281
ttttttttcg tgggaaggaa aatgcttctg ccaggcagat gccatatgcg ccgtgtactc


 


2341
aggtttttcc tatttattgt ggactggaag ctcgccatct ccgcccggca gaccgggcag


 


2401
atccggcatg ggctggcacc cggggcctta agaactcctg ctctcttgcc acaacgcttt


 


2461
tgtctcctcg ctatctgaat ggcaccctcc ttctccctca ctctctccat cccattctct


 


2521
gcattctctt ggttttctct cccttttgct ttgtcgctga cacccctgcc caccccatgc


 


2581
tggccctgtt tctctcctgc ccctccctcc ccagctctcc atccctcacc ctctgtgctt


 


2641
ctgtctccat ccctggctct ccagcgtccc tggccttttg gtccctgagc tttaatgcct


 


2701
ttccctgcct tctgttctta tttggactgc agtggccctt tgcaggagct ctggaggccc


 


2761
aggggctgag gaggagggtt acccctctac ccatctgaaa cctagggtct agggggatca


 


2821
aggaaaaaaa gtccccaaag aaggggaatt ttttgtttgt ttttgagggg agatcccaga


 


2881
aatgtagctt gtttcatatt ttagtcttct tatttttgta aaatgtgtag aatttgctgt


 


2941
ttttcttttt cttttgacaa ctcaggaaga aactgacctc agaaagaatg ttagactttg


 


3001
gctgctctcc tgtgtgcccc tcacacctgc cccctccccc ccactccatc caggggacca


 


3061
aattctccca gacactcaaa aaatgagact tacggggaag gggagaggaa gacccagagg


 


3121
cctcagtgaa accccagcta ttcctggtca gaagcagaat gtattcctaa gggcttcctc


 


3181
cccagggccg aggcctaggc atgaatgtgg ggagtgggct gtggggtttg agagaaggga


 


3241
ggccttattc ctctcctgct gctccccacc ccctgcccca cccaacccct ccgctgagtg


 


3301
ttttctgtga agggctatcc agagttagga tgcccttgcc caattccttc ctgagaccca


 


3361
gaaggtaggg tgggagggcc caaatgggaa ggtgacctaa gcagaaagtc tccagaaagg


 


3421
tcatgtcccc tggccctgcc ttggcagagg tccccagtga cttatgctag gaggattcca


 


3481
tctgggtaga cagtctggcc acaaaatcag ctactggacc tcagccatct ctgctggagg


 


3541
ctctgaggag gagtgagcat ccctcacttg tgggggctct gtgaggaaat gtgccttccc


 


3601
cattcccccg gagtcctagg tctggagctc cagggctggg agagggtgag ggagatgggc


 


3661
aggggtgttt tctctgacct tgggggctta gtctcagtcc tgcctgaact ttccactagg


 


3721
cttggaaccc ttccaagaac catatttctc tccttcccac caattttccc ttgatgaggc


 


3781
tttagcagtt tgctcccacc acccccagcc catttcacaa ctctgatctt agtccaaagc


 


3841
aggggacacg cccccccacc accacttttt ctctctccca tctcagcctc ctgtgcagtt


 


3901
ccttgcctgc ccgtgcattt cctagagtct actgcctccc ccctggctgg gagggtgtct


 


3961
gggggggatc tttcaggggc cctggcaccc agggcctgtg ctggcctagg agtgctgacc


 


4021
agaaggctgc tctgttcccc cccacccccg ttgctttctg gccccctctt tggagccagc


 


4081
cacccacagg gctttggtgc ctcagaagca gtgggctgcc gggtcacagc cgcaggctgc


 


4141
aaaagaccct cggagggagc atggagtgag gggttctctc tcaggtgtgt atgtattggg


 


4201
gggtgggggt gggtggaggg tgtcagggaa gttggggtgg gatcccagcc ttcccttcaa


 


4261
gaggcaggga gctctgggag gtggagtccc caccgctttc tctactaggc tcctcctgtt


 


4321
ccccaggctt ggggagcttt gcacaaggag actgccccca gcctagtggc acctacctca


 


4381
tgggctctgg ggcaggtagg ggaagggcca gtccagctct ggtaatgctg gggggaggca


 


4441
taccaaagaa tccaggggca gggagtgggg agggtgactt ccgagctggc ctctcccctt


 


4501
cctctaccca gactggggct gggatcctct cctcccgctg taaccatttc tacctcattt


 


4561
tgctgcgtgt tgtacatgga cgtatttatc tcctgtctga cgatgctctg cagttgtggt


 


4621
ctgtctacct cagaagagac tgtattttaa aagaaagtat tacacagtat taaagcgatg


 


4681
acatgtggtt tgcaaaaaaa aaaaaaaaaa a






which encodes:








(human Fidgetin-like 2)


(SEQ ID NO: 2)


MHWTPEHAQPLNQWPEQHLDVSSTTPSPAHKLELPPGGRQRCHYAWA


 


HDDISALTASNLLKRYAEKYSGVLDSPYERPALGGYSDASFLNGAKG


 


DPEPWPGPEPPYPLASLHEGLPGTKSGGGGGSGALGGSPVLAGNLPE


 


PLYAGNACGGPSAAPEYAAGYGGGYLAPGYCAQTGAALPPPPPAALL


 


QPPPPPGYGPSAPLYNYPAGGYAAQPGYGALPPPPGPPPAPYLTPGL


 


PAPTPLPAPAPPTAYGFPTAAPGAESGLSLKRKAADEGPEGRYRKYA


 


YEPAKAPVADGASYPAADNGECRGNGFRAKPPGAAEEASGKYGGGVP


 


LKVLGSPVYGPQLEPFEKFPERAPAPRGGFAVPSGETPKGVDPGALE


 


LVTSKMVDCGPPVQWADVAGQGALKAALEEELVWPLLRPPAYPGSLR


 


PPRTVLLFGPRGAGKALLGRCLATQLGATLLRLRGATLAAPGAAEGA


 


RLLQAAFAAARCRPPSVLLISELEALLPARDDGAAAGGALQVPLLAC


 


LDGGCGAGADGVLVVGTTSRPAALDEATRRRFSLRFYVALPDSPARG


 


QILQRALAQQGCALSERELAALVQGTQGFSGGELGQLCQQAAAGAG


 


LPGLQRPLSYKDLEAALAKVGPRASAKELDSFVEWDKMYGSGH.






In embodiments, the siRNA comprise one of the following pairs of sense/antisense sequences:









Sense:



(SEQ ID NO: 3)



UUACACAGUAUUAAAGCGAUU 



 



Antisense:



(SEQ ID NO: 4)



5′ UCGCUUUAAUACUGUGUAAUU;



or



 



Sense:



(SEQ ID NO: 5)



CAUCUGAAACCUAGGGUCUUU 



 



Antisense:



(SEQ ID NO: 6)



5′ AGACCCUAGGUUUCAGAUGUU;



or



 



Sense:



(SEQ ID NO: 7)



GUGACUUAUGCUAGGAGGAUU 



 



Antisense:



(SEQ ID NO: 8)



5′ UCCUCCUAGCAUAAGUCACUU;



or



 



Sense:



(SEQ ID NO: 9)



GGUCAGAAGCAGAAUGUAUUU 



 



Antisense:



(SEQ ID NO: 10)



5′ AUACAUUCUGCUUCUGACCUU.






In an embodiment, the siRNA is double-stranded and comprises SEQ ID NO:3 and 4; SEQ ID NO:5 and 6; SEQ ID NO:7 and 8; or SEQ ID NO:9 and 10.
In an embodiment, the 5′ terminal residue of a strand of the siRNA is phosphorylated. In an embodiment the 5′ terminal residue of the antisense strand of the siRNA is phosphorylated.
As used herein an “aptamer” is a single-stranded oligonucleotide or oligonucleotide analog that binds to a particular target molecule, such as a Fidgetin-like 2 protein, or to a nucleic acid encoding a Fidgetin-like 2 protein, and inhibits the function or expression thereof, as appropriate. Alternatively, an aptamer may be a protein aptamer which consists of a variable peptide loop attached at both ends to a protein scaffold that interferes with Fidgetin-like 2 protein interactions
The present invention provides kits for treating wounds, preferably skin wounds.
A composition provided in such a kit may be provided in a form suitable for reconstitution prior to use (such as a lyophilized injectable composition) or in a form which is suitable for immediate application to a wound, including to the wound margin, such as a lotion or ointment.
In an embodiment of the invention the inhibitor of fidgetin-like 2 is provided by a subcutaneous implant or depot medicament system for the pulsatile delivery of the inhibitor to a wound or site where a wound is to expected be formed to promote wound healing. The inhibitor can be provided, for example, in a therapeutically effective amount to each centimeter of a wound margin or each centimeter of a site at which a wound is expected to be formed.
A medicament in accordance with this aspect of the invention may be formulated in any appropriate carrier. Suitable carriers are pharmaceutically acceptable carriers, preferably those consistent with administration topically or administration by injection.
It will be appreciated that, while the inhibitor of Fidgetin-like 2 may be administered by the same route and in the same form in each incidence of treatment, different incidences of treatment may provide the inhibitor of Fidgetin-like 2 by different medicaments and/or different routes of administration. In embodiments of the invention the initial incidence of treatment may provide the inhibitor of Fidgetin-like 2 by means of an injection, such as an intradermal injection, while the second (and any subsequent) incidences of treatment may involve provision of the inhibitor of Fidgetin-like 2 by alternative routes, such as topical formulations, or vice versa. In an embodiment, multiple administrations of the inhibitor of Fidgetin-like 2 may be effected by the same means or route.
The benefits that may be derived from the present invention may be applicable to wounds at sites throughout the body. However, it may be preferred that the wound for which healing is promoted is a skin wound. For illustrative purposes the embodiments of the invention will generally be described with reference to skin wounds, although they remain applicable to other tissues and organs. Merely by way of example, in another preferred embodiment the wound may be a wound of the circulatory system, particularly of a blood vessel. Other wounds in which wound healing may be promoted in accordance with the present invention include as a result of surgery or as a result of a burn. Other wounds in which wound healing may be promoted in accordance with the present invention include skin ulcers caused by pressure, venous stasis, or diabetes mellitus.
In a non-limiting embodiment the inhibitor of Fidgetin-like 2 is provided in a bulk-eroding system such as polylactic acid and glycolic acid (PLGA) copolymer based microspheres or microcapsules systems containing the inhibitor of Fidgetin-like 2. In an embodiment, blends of PLGA:ethylcellulose systems may be used as an appropriate carrier. A further medicament in accordance with this aspect of the invention may be formulated in a surface-eroding system wherein the inhibitor of Fidgetin-like 2 is embedded in an erodible matrix such as the poly(ortho) ester and polyanhydride matrices wherein the hydrolysis of the polymer is rapid. A medicament in accordance with this aspect of the invention may also be formulated by combining a pulsatile delivery system as described above and an immediate release system such as a lyophilized injectable composition described above.
Examples of specific wounds in which healing may be promoted using the medicaments and methods of the invention include, but are not limited to, those independently selected from the group consisting of: wounds of the skin; wounds of the eye (including the inhibition of scarring resulting from eye surgery such as LASIK surgery, LASEK surgery, PRK surgery, glaucoma filtration surgery, cataract surgery, or surgery in which the lens capsule may be subject to scarring) such as those giving rise to corneal cicatrisation; wounds subject to capsular contraction (which is common surrounding breast implants); wounds of blood vessels; wounds of the central and peripheral nervous system (where prevention, reduction or inhibition of scarring may enhance neuronal reconnection and/or neuronal function); wounds of tendons, ligaments or muscle; wounds of the oral cavity, including the lips and palate (for example, to inhibit scarring resulting from treatment of cleft lip or palate); wounds of the internal organs such as the liver, heart, brain, digestive tissues and reproductive tissues; wounds of body cavities such as the abdominal cavity, pelvic cavity and thoracic cavity (where inhibition of scarring may reduce the number of incidences of adhesion formation and/or the size of adhesions formed); and surgical wounds (in particular wounds associated with cosmetic procedures, such as scar revision). It is particularly preferred that the medicaments and methods of the invention be used to promote healing of wounds of the skin.
The inhibitor may be used in a composition with additives. Examples of suitable additives are sodium alginate, as a gelatinizing agent for preparing a suitable base, or cellulose derivatives, such as guar or xanthan gum, inorganic gelatinizing agents, such as aluminum hydroxide or bentonites (termed thixotropic gel-formers), polyacrylic acid derivatives, such as Carbopol®, polyvinylpyrrolidone, microcrystalline cellulose and carboxymethylcellulose. Amphiphilic low molecular weight and higher molecular weight compounds, and also phospholipids, are also suitable. The gels can be present either as water-based hydrogels or as hydrophobic organogels, for example based on mixtures of low and high molecular weight paraffin hydrocarbons and vaseline. The hydrophilic organogels can be prepared, for example, on the basis of high molecular weight polyethylene glycols. These gelatinous forms are washable. Hydrophobic organogels are also suitable. Hydrophobic additives, such as petroleum jelly, wax, oleyl alcohol, propylene glycol monostearate and/or propylene glycol monopalmitostearate, in particular isopropyl myristate can be included. In an embodiment the inhibitor is in a composition comprising one or more dyes, for example yellow and/or red iron oxide and/or titanium dioxide for the purpose of matching as regards color. Compositions may be in any suitable form including gels, lotions, balms, pastes, sprays, powders, bandages, wound dressing, emulsions, creams and ointments of the mixed-phase or amphiphilic emulsion systems (oil/water-water/oil mixed phase), liposomes and transfersomes or plasters/band aid-type coverings. Emulsifiers which can be employed in compositions comprising the inhibitor of Fidgetin-like 2 include anionic, cationic or neutral surfactants, for example alkali metal soaps, metal soaps, amine soaps, sulphonated and sulphonated compounds, invert soaps, higher fatty alcohols, partial fatty acid esters of sorbitan and polyoxyethylene sorbitan, e.g. lanette types, wool wax, lanolin or other synthetic products for preparing the oil/water and/or water/oil emulsions.
Compositions comprising the inhibitor of Fidgetin-like 2 can also comprise vaseline, natural or synthetic waxes, fatty acids, fatty alcohols, fatty acid esters, for example as monoglycerides, diglycerides or triglycerides, paraffin oil or vegetable oils, hydrogenated castor oil or coconut oil, hog fat, synthetic fats (for example based on caprylic acid, capric acid, lauric acid or stearic acid, such as Softisan®), or triglyceride mixtures, such as Miglyol®, can be used as lipids, in the form of fatty and/or oleaginous and/or waxy components for preparing the ointments, creams or emulsions of the compositions comprising the inhibitor of fidgetin-like 2 used in the methods described herein.
Osmotically active acids and alkaline solutions, for example hydrochloric acid, citric acid, sodium hydroxide solution, potassium hydroxide solution, sodium hydrogen carbonate, may also be ingredients of the compositions and, in addition, buffer systems, such as citrate, phosphate, tris buffer or triethanolamine, for adjusting the pH. It is possible to add preservatives as well, such as methyl benzoate or propyl benzoate (parabens) or sorbic acid, for increasing the stability.
Pastes, powders and solutions are additional forms of compositions comprising the inhibitor of Fidgetin-like 2 which can be applied topically. As consistency-imparting bases, the pastes frequently contain hydrophobic and hydrophilic auxiliary substances, preferably, however, hydrophobic auxiliary substances containing a very high proportion of solids. In order to increase dispersity, and also flowability and slipperiness, and also to prevent agglomerates, the powders or topically applicable powders can, for example, contain starch species, such as wheat or rice starch, flame-dispersed silicon dioxide or siliceous earth, which also serve as diluent.
In an embodiment, the compositions comprise further active ingredients suitable for protecting or aiding in healing of the wound, for example one or more antibiotics, antiseptics, vitamins, anesthetics, antihistamines, anti-inflammatory agents, moisturizers, penetration-enhancing agents and/or anti-irritants.
In an embodiment of the methods and compositions described herein the subject is a mammal. In an embodiment the subject is human.
As used herein, “promotion” of wound healing, or grammatical equivalent, means an acceleration in any one or more of visual appearance of wound recovery, reduction in wound size, reduction in distance between wound margins, scab formation, fibroplasia and re-epithelialization as compared to the corresponding parameter in an untreated wound.
As used herein, “wound” is a break or discontinuity in the structure of an organ or tissue (including skin), which includes epithelium, connective tissue, and muscle tissue, caused by an external agent. Examples of wounds include, but are not limited to, skin wounds, ulcerations, bedsores, grazes, tears, cuts, punctures, tympanic membrane perforations, burns, and those that are a consequence of plastic surgery procedures.
With regard to the methods described herein to identify candidate agents as inhibitors of Fidgetin-like 2, one skilled in the art can readily screen libraries of compounds, for example small molecule libraries, using the methods as described to identify agents which are inhibitors of Fidgetin-like 2 and which are therapeutic in treating wounds and promoting the healing of wounds. In addition, one skilled in the art can employ the method to identify peptides, peptidomimetics, antibodies, antibody fragments and nucleic acids which are inhibitors of Fidgetin-like 2 and which are therapeutic in treating wounds and promoting the healing of wounds.
The method can be employed as an assay using detection and quantification techniques known in the art, including those pertaining to measuring enzyme activity, such as the ATPase activity of Fidegtin-like 2.
The methods can be used to identify inhibitors of Fidgetin-like 2 which can then be applied to wound models to determine if the agent promotes/accelerates wound healing, especially for skin.
Preferably the inhibitor is biomembrane-permeable or is conjugated or otherwise attached to a moiety which renders the inhibitor biomembrane-permeable.
A method is also provided for treating wound in a subject comprising administering to the subject an amount of an inhibitor of Fidgetin effective to treat the wound. In an embodiment, the inhibitor of Fidgetin is a nucleic acid. In an embodiment, the inhibitor of Fidgetin is an siRNA or shRNA. In an embodiment, the nucleic acid is directed against a DNA or mRNA encoding Fidgetin. In an embodiment, the Fidgetin is human Fidgetin. In an embodiment, the wound is a neuronal wound. In an embodiment, the inhibitor of Fidgetin is encapsulated in a nanoparticle. In an embodiment, the nanoparticle is a liposomal nanoparticle. In an embodiment, the Fidgetin comprises the following sequence:







(SEQ ID NO: 11)








  1
MISSTSVYGL KMQWTPEHAQ WPEQHFDITS TTRSPAHKVE



AYRGHLQRTY QYAWANDDIS


 


 61
ALTASNLLKK YAEKYSGILE GPVDRPVLSN YSDTPSGLVN



GRKNESEPWQ PSLNSEAVYP


 


121
MNCVPDVITA SKAGVSSALP PADVSASIGS SPGVASNLTE



PSYSSSTCGS HTVPSLHAGL


 


181
PSQEYAPGYN GSYLHSTYSS QPAPALPSPH PSPLHSSGLL



QPPPPPPPPP ALVPGYNGTS


 


241
NLSSYSYPSA SYPPQTAVGS GYSPGGAPPP PSAYLPSGIP



APTPLPPTTV PGYTYQGHGL


 


301
TPIAPSALTN SSASSLKRKA FYMAGQGDMD SSYGNYSYGQ



QRSTQSPMYR MPDNSISNTN


 


361
RGNGFDRSAE TSSLAFKPTK QLMSSEQQRK FSSQSSRALT



PPSYSTAKNS LGSRSSESFG


 


421
KYTSPVMSEH GDEHRQLLSH PMQGPGLRAA TSSNHSVDEQ



LKNTDTHLID LVTNEIITQG


 


481
PPVDWNDIAG LDLVKAVIKE EVLWPVLRSD AFSGLTALPR



SILLFGPRGT GKTLLGRCIA


 


541
SQLGATFFKI AGSGLVAKWL GEAEKIIHAS FLVARCRQPS



VIFVSDIDML LSSQVNEEHS


 


601
PVSRMRTEFL MQLDTVLTSA EDQIVVICAT SKPEEIDESL



RRYFMKRLLI PLPDSTARHQ


 


661
IIVQLLSQHN YCLNDKEFAL LVQRTEGFSG LDVAHLCQEA



VVGPLHAMPA TDLSAIMPSQ


 


721
LRPVTYQDFE NAFCKIQPSI SQKELDMYVE WNKMFGCSQ.






In an embodiment, the Fidgetin is encoded by a nucleic acid sequence comprising the following:








(SEQ ID NO: 12)










   1
gggtttgaaa ttccaacatg gcagaggctg cagtccgtct tcccttcaaa aacttggaat



 


  61
gatttcaaat cataggcacc ttcacttaac cctagcttcc attcatcagc aaacacatcg


 


 121
gatcgatgct acgctaacct atcgggttct ctctccgcgc gttcaggtta aatgaatacc


 


 181
tgacgaaagg gcccacgttt caaggcagtg acatttgata gctgagagga aaagtggctt


 


 241
taatgaaaag caacctttgg aattcctgct tgtgagaaat ccaattcagc tttttgtgct


 


 301
gccagcaaga aatgatcagt agcaccagtg tttatggctt gaagatgcag tggacgccag


 


 361
agcatgccca gtggccagaa cagcactttg acatcacctc aaccactcgg tctcctgccc


 


 421
acaaagttga agcctacaga ggtcatctgc agcgcaccta tcagtacgcc tgggcgaatg


 


 481
atgacatatc tgctctgact gcatccaacc tactaaaaaa atatgcagag aagtattccg


 


 541
gcattttgga aggtcctgtg gaccgacccg tactcagcaa ctattcggac acaccatcag


 


 601
gactagtgaa cggtcggaaa aatgaaagtg aaccctggca gccttccttg aattcagaag


 


 661
ctgtttatcc catgaactgt gttccggatg ttatcactgc cagcaaagct ggagtcagtt


 


 721
cagccctccc tccagcagat gtctctgcga gtataggaag ctctcctggg gtagccagca


 


 781
acctgacaga acctagttat tcaagtagta cctgtggaag ccacactgta cccagtcttc


 


 841
atgcagggct cccatctcag gaatatgccc caggatacaa cggatcatat ttgcattcta


 


 901
cttatagtag ccagccagca cctgcacttc cttcacctca tccgtctcct ttgcatagct


 


 961
ctgggctact acagccccca ccaccacctc ctccgccacc agccttggtc ccaggctaca


 


1021
atgggacttc taacctctcc agttacagct atccgtctgc tagctatcct cctcagactg


 


1081
ctgtggggtc tgggtacagc cctggggggg caccgcctcc gccttcagcg tacctgcctt


 


1141
caggaattcc tgctcccacc cccctacccc ccaccactgt tcctggctac acctaccagg


 


1201
gccatggttt gacacctatt gcaccgtcgg ctctgacaaa cagttcagca agttctctca


 


1261
aaaggaaagc tttctacatg gcagggcaag gagatatgga ctccagttat ggaaattaca


 


1321
gctatggcca acagagatct acacagagtc ctatgtacag aatgcccgac aacagcattt


 


1381
caaacacaaa tcgggggaat ggctttgaca gaagtgctga aacatcatcc ttagcattta


 


1441
agccaacgaa gcagctaatg tcctctgaac agcaaaggaa attcagcagc cagtccagta


 


1501
gggctctgac ccctccttcc tacagtactg ctaaaaattc attgggatca agatccagtg


 


1561
aatcctttgg gaagtacaca tcgccagtaa tgagtgagca tggggacgag cacaggcagc


 


1621
tcctctctca cccaatgcaa ggccctggac tccgtgcagc tacctcatcc aaccactctg


 


1681
tggacgagca actgaagaat actgacacgc acctcatcga cctggtaacc aatgagatta


 


1741
tcacccaagg acctccagtg gactggaatg acattgctgg tctcgacctg gtgaaggctg


 


1801
tcattaaaga ggaggtttta tggccagtgt tgaggtcaga cgcgttcagt ggactgacgg


 


1861
ccttacctcg gagcatcctt ttatttggac ctcgggggac aggcaaaaca ttattgggca


 


1921
gatgcatcgc tagtcagctg ggggccacat ttttcaaaat tgccggttct ggactagtcg


 


1981
ccaagtggtt aggagaagca gagaaaatta tccatgcctc ttttcttgtg gccaggtgtc


 


2041
gccagccctc ggtgattttt gttagtgaca ttgacatgct tctctcctct caagtgaatg


 


2101
aggaacatag tccagtcagt cggatgagaa ccgaatttct gatgcaactg gacactgtac


 


2161
taacttcggc tgaggaccaa atcgtagtaa tttgtgccac cagtaaacca gaagaaatag


 


2221
atgaatccct tcggaggtac ttcatgaaac gacttttaat cccacttcct gacagcacag


 


2281
cgaggcacca gataatagta caactgctct cacagcacaa ttactgtctc aatgacaagg


 


2341
agtttgcact gctcgtccag cgcacagaag gcttttctgg actagatgtg gctcatttgt


 


2401
gtcaggaagc agtggtgggc cccctccatg ccatgccagc cacagacctt tcagccatta


 


2461
tgcccagcca gttgaggccc gttacatatc aagactttga aaatgctttc tgcaagattc


 


2521
agcctagcat atctcaaaag gagcttgata tgtatgttga atggaacaaa atgtttggtt


 


2581
gcagtcagtg ataacttctt tagaaaaaaa aaatgtaatg aatgttggca cacacacata


 


2641
aaacctgcta catagggaat agagcccctt tecagtagag tttaaattgc aaagggtact


 


2701
ggggaagatg acgattaagt tgcatcttta gagtcagggt agatttggag gaaaagtgca


 


2761
tcaaatgaga gcttctgatt tgaaagcccc agatgacaga aagcatatgt ggatgctcag


 


2821
ttctgttcaa gctagacaac actcaccaag gagcaaggtg caagtgtgtt gatttcagaa


 


2881
ggacatgaac ctcgtgtgtt gattccattc tgctgttctc gagatttagt tgctgtcaag


 


2941
tgcctggagt ggtgctttat tttttgtttg cctcacaatt acattggtgg catgtgctaa


 


3001
tataaagagc tttaacttca aacattattg gactaaagag atgaacagtt gtgttatgac


 


3061
agaaaaccag atttttgcca ttttaagagc aacagtattc ctcaatcctg tctgttctgc


 


3121
agtattaagc taagaacagg taaaacaggg taacggtaat ctggacctta atttctgcag


 


3181
ttcatttctt ttaatgttct tgtctgcaaa aactcaggaa agtgattgtg atttgtacag


 


3241
tacctcaaag gaatgtgttg aaagcactat gtactgctga gagtaatagg ataggcttca


 


3301
atgttacttt atattaaaat gtatgtttac ctcaacaatt ggaaaatagc aaggaaaatt


 


3361
actttgaatg tatccagaaa aatactgaag tgtgatacaa ctgaatattt acagtttaaa


 


3421
gtagaaatgg aaggattttt ttaagttctt ttactaatta tggggaatta accagagcag


 


3481
aataattctt tatgtcaata actgcaagag ttcttagtac attgctcctt gataattaag


 


3541
tgaaaatgtt cttaaaaggt acactggtta attgaaagct acttattcag tttgtgttag


 


3601
tgtctagacc tgtcagccac aagacctgtt taggaccctg aaagtcacag tacctaaaaa


 


3661
ctatgactgc ctttttattg cataggtggt agtggtggtg atggtggtgg tagtttgcaa


 


3721
gttatctctt aaaactgctg ggaatggtgt cattctattc actaatctag cttatagact


 


3781
tgccgtgctg tttgatagaa tgcagaggat agcaaccaaa acaaatacac aaataaataa


 


3841
aaacaaaaac caaccaacaa accaacttac atacacatat atatatccac aaagaacctc


 


3901
tccatctcct ccccttcttt ttgactccac tcttgtcagt gcaattttgc ttctcatttt


 


3961
gaaatctggg ctgtagtgct cctgctttat ttctacctca gttttgttac atttctcttg


 


4021
gaaagtaaag tagaaaattg gaagtggaca cacacactgc aatgtagctt gccaaacatg


 


4081
ttactttgtt ttcttccatc tttcaccgta aatctagttt ccaaagacat cagcatttgt


 


4141
gcttacttcc acctcagtct accagcccca cccctaccca tggcataagt ggcatttttc


 


4201
ttaatttcct atttttctcc tgctctctgt caagttgttc tttgtatcct ttaatgcttt


 


4261
atgtgcaacc tttcattgat agtgggctga tgtttggcaa tgcttctgaa ctgtcacaga


 


4321
gcaggctgta gctttccaca gccactgccc atgcataagc agaacagcct ggccttttga


 


4381
atgtattttc ctgggttttt tccccttttc tttttttagt ttagagatgc agtaacaaaa


 


4441
ctgttgcaaa gcactggcat tttatgtatt caataaataa gtgatgtaca tttttaaaaa


 


4501
aatttaaata aatgcaatga gaagccccaa gaaag






All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
EXPERIMENTAL DETAILS
Introduction
At present, papers have not been published on human Fidgetin-like 2, but the mouse homologue has been found to be highly expressed in most tissues (with the exception of testes) (Yang, Mahaffey et al. 2005). However, this laboratory has now identified the following role for human Fidgetin-like 2.
Results
Fidgetin-like 2 is expressed in human tissue culture cells where it localizes to microtubules. FIG. 1A shows a Western blot of human U2OS cell lysates probed with an anti-Fidgetin-like 2 antibody generated in lab. This antibody recognizes a single band that is substantially decreased by Fidgetin-like 2 siRNA treatment (see FIG. 2 below). FIG. 1B shows a migrating U2OS cell double-labeled for Fidgetin-like 2 and microtubules. At high magnification (inset), Fidgetin-like 2 clearly co-localizes with spans of the microtubule lattice near the cell edge.
Cells depleted of Fidgetin-like 2 display a several-fold increase in their rate of wound healing and migration (FIG. 2). FIG. 2A shows Western blots of U2OS cell lysates obtained from control (N) and Fidgetin-like 2 siRNA treated cultures (72 hrs after treatment). Actin was used as a loading control. FIG. 2B shows time-lapse phase contrast images of “wound healing” assays performed in control and Fidgetin-like 2 siRNA treated cultures. In these assays, a monolayer of U2OS cells is “wounded” by a pipette tip and the invasion of cells into the wound is monitored over time. FIG. 2C shows the average rate of wound closure in each condition which is increased nearly 4-fold after Fidgetin-like 2 siRNA. FIG. 2D shows the trajectories of single control and Fidgetin-like 2 siRNA treated cells as they enter the wound zone. Not only do Fidgetin-like 2 siRNA-treated cells move several-fold faster than controls, they also display more directionally persistent migration as indicated in FIG. 2E.
Fidgetin-like 2 siRNA also dramatically enhances chemotaxis of cultured human cells (FIG. 3). The impact of Fidgetin-like 2 siRNA on chemotaxis of human U2OS cells was measured in a transwell assay (modified Boyden chamber). This assay counts the number of cells that move through 8 μm pores towards a chemoattractant loaded in the distal well. The graph in FIG. 3 shows the number of control (N) and siRNA-treated (F12) cells that migrated through the pores before and three hours after the addition of a chemoattractant (Fetal Bovine Serum).
Fidgetin-like 2 siRNA elevates the rate of U2OS cell proliferation (FIG. 4). Although the biochemical activity of Fidgetin-like 2 has not been previously demonstrated, we have found that the closely related protein, Fidgetin, utilizes ATP hydrolysis to induce microtubule severing and depolymerization, in vitro (FIG. 5). FIG. 5 shows kymographs showing (left panel) severing of a MT incubated 50 nM recombinant Fidgetin and (right panel) depolymerization at the minus-end of a polarity marked induced MT incubated with 25 nM Fidgetin. ATP was added in both conditions. The reaction is entirely inhibited by the non-hydrolyzable ATP analogue, AMPPNP
The studies of Fidgetin-like 2 were repeated in human dermal fibroblasts (adult). Fibroblasts depleted of Fidgetin-like 2 displayed a >2-fold increase in the rate of “wound closure” as determined by a standard scratch assay (see FIG. 6). The left panels of FIG. 6 show time-lapse phase contrast images of “wound healing” assays performed in control and Fidgetin-like 2 siRNA-treated cultures of human dermal fibroblasts. In these assays, a monolayer is “wounded” by a pipette tip and the invasion of cells into the wound is monitored over time. The right panel of FIG. 6 shows the average rate of wound closure measured in each condition.
In further experiments, topical application of Fidgetin-like 2 siRNA (encapsulated in nanoparticles) to mouse full thickness biopsy wounds was found to enhance wound healing (FIGS. 7, 8 and 9). In addition, depletion of Fidgetin-like 2 from embryonic mouse hearts was found to stimulate neoangiogenesis (FIG. 10). Furthermore, depletion of Fidgetin, and depletion of Fidgetin-like 2 from rat primary hippocampal neurons were both found to promote axon regrowth (FIGS. 11, 12 and 13).
EXAMPLE
A skin wound in a human subject is treated with a topically applied siRNA or shRNA which inhibits Fidgetin-like 2. The topically applied siRNA or shRNA is effective to treat the skin wound in the human subject. The topically applied siRNA or shRNA accelerates skin wound healing in the human subject.
REFERENCES


    Yang, Y., C. L. Mahaffey, et al. (2005). “Functional characterization of fidgetin, an AAA-family protein mutated in fidget mice.” Exp Cell Res 304(1): 50-58.

## Claims

1. A method of reducing a scar comprising directly administering to the scar an amount of a siRNA or shRNA directed against a DNA or RNA encoding a human Fidgetin like-2 comprising the amino acid set forth in SEQ ID NO:2 effective to reduce a scar.

2. A method of stimulating neoangiogenesis in a tissue comprising directly administering to the tissue an amount of a siRNA or shRNA directed against a DNA or RNA encoding a human Fidgetin like-2 comprising the amino acid set forth in SEQ ID NO:2 effective to stimulate neoangiogenesis in a tissue.