WO2015153880A2 - Modulation of hotair and adipogenesis - Google Patents

Abstract

Provided herein are compositions and methods for forming adipose tissue. One aspect provides a method including contacting adipose progenitor cells and an agent for modulating HOX antisense intergenic RNA ("HOTAIR"), an adipose-derived exosome, a miRNA associated with an adipose-derived exosome, or a demethylation agent. Such contact can induce or increase adipogenesis, formation of white adipose, formation brown adipose, or transition of white adipose to brown adipose.

Description

TITLE OF INVENTION

MODULATION OF HOTAIR AND ADIPOGENESIS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Patent

Application Serial No. 62/052,868 filed 19 September 2014 and U.S. Provisional Patent Application Serial No. 61/974,361 filed 02 April 2014, each of which is incorporated herein by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is

incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Soft tissue craniofacial defects resulting from surgery, trauma, or congenital malformations propose serious esthetic challenges for patients that may incite psychosocial distress. Currently, one of the most widespread methods to address these defects in plastic surgery is autologous fat grafting, which improves tissue volume and facial contour. But clinical outcomes are

unpredictable and include problems such as donor site morbidity, graft necrosis with subsequent fibrosis, or graft integration failure. Further, obesity and adipose tissue-related diseases have rapidly grown in prevalence in recent years. Gene therapy is a highly sought after therapeutic approach for the potential to alter cellular function in vivo, but gene therapy has traditionally used viral vectors that have issues with immunogenicity.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision of compositions and methods for forming adipose tissue. One aspect provides a method of forming adipose tissue in a subject in need thereof. In some embodiments, the method includes contacting an adipose tissue or an adipose progenitor cell of a subject and a first composition comprising HOX antisense intergenic RNA ("HOTAIR") in an amount effective to increase formation of white adipose tissue or cells. In some embodiments, the method includes contacting an adipose tissue or an adipose progenitor cell of a subject and a second composition comprising a nucleic acid construct encoding HOTAIR, wherein the nucleic acid construct transforms the adipose progenitor cell so as to increase expression of HOTAIR in an amount effective to increase formation of white adipose tissue or cells. In some embodiments, the method includes contacting an adipose tissue or an adipose progenitor cell of a subject and a third composition comprising an RNA interference molecule specific for HOTAIR, wherein the RNA interference molecule decreases expression of HOTAIR by an amount effective to increase formation of brown adipose tissue or cells. In some embodiments, the method includes contacting an adipose tissue or an adipose progenitor cell of a subject and a fourth composition comprising an adipose-derived exosome in an amount effective to increase formation of white or brown adipose tissue or cells. In some embodiments, the method includes contacting an adipose tissue or an adipose progenitor cell of a subject and a fifth composition comprising one or more miRNA selected from the group consisting of miR210 (SEQ ID NO: 21 ), miR296 (SEQ ID NO: 22), miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25), or a miRNA having at least about 95% sequence identity thereto and retaining an activity associated with the miRNA, in an amount effective to increase formation of white or brown adipose tissue or cells. In some embodiments, the method includes contacting an adipose tissue or an adipose progenitor cell of a subject and a sixth composition comprising a demethylation agent in an amount effective to increase formation of brown adipose tissue or cells.

Another aspect provides a composition comprising an adipose formation or modulation agent. In some embodiments, the agent includes a first

composition comprising HOX antisense intergenic RNA ("HOTAIR") in an amount effective to increase formation of white adipose tissue or cells. In some embodiments, the agent includes a second composition comprising a nucleic acid construct encoding HOTAIR, wherein the nucleic acid construct transforms the adipose progenitor cell so as to increase expression of HOTAIR in an amount effective to increase formation of white adipose tissue or cells. In some embodiments, the agent includes a third composition comprising a protein aptamer, nucleotide aptamer, or RNA interference molecule specific for HOTAIR, wherein the third composition decreases expression of HOTAIR by an amount effective to increase formation of brown adipose tissue or cells. In some embodiments, the agent includes a fourth composition comprising an adipose- derived exosome in an amount effective to increase formation of white or brown adipose tissue or cells. In some embodiments, the agent includes a fifth composition comprising one or more miRNA selected from the group consisting of miR210 (SEQ ID NO: 21 ), miR296 (SEQ ID NO: 22), miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25), or a miRNA having at least about 95% sequence identity thereto and retaining an activity associated with the miRNA, in an amount effective to increase formation of white or brown adipose tissue or cells. In some embodiments, the agent includes a sixth composition comprising a demethylation agent in an amount effective to increase formation of brown adipose tissue or cells.

In some embodiments, a composition above, or component thereof, can be operably linked to a nanoparticle.

In some embodiments, the first composition comprises SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 95% sequence identity thereto, or a functional fragment thereof, having an activity associated with HOTAIR.

In some embodiments, the second composition comprises an expression construct having a promoter operably linked to a transcribable nucleic acid molecule comprising SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 95% sequence identity thereto, or a functional fragment thereof, having an activity associated with HOTAIR.

In some embodiments, the third composition comprises a protein aptamer, nucleotide aptamer, or RNA interference molecule specific for SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 95% sequence identity thereto, or a fragment thereof. In some embodiments, the third composition comprises a protein aptamer, nucleotide aptamer, or RNA interference molecule specific for SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In some embodiments, the RNA interference molecule comprises a small interfering RNA (siRNA), short hairpin RNA

(shRNA), or micro RNA (miRNA).

In some embodiments, the sixth composition comprises a demethylation agent selected from thr group consisting of (i) 5-Aza-2'-deoxycytidine, (ii) 5- azacytidine, or (iii) an antisense oligonucleotide, protein aptamer, nucelotide aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), or micro RNA (miRNA) specific for a target DNA methyltransferase.

In some embodiments, contacting adipose tissue or an adipose progenitor cell occurs in vivo. In some embodiments, the adipose progenitor cells comprise a CD31 -CD34+CD146- subpopulation of adipose progenitor cells. In some embodiments, contacting adipose tissue or an adipose progenitor cell occurs ex vivo; and the method further comprises transplanting the contacted adipose progenitor cells into a subject in need thereof.

In some embodiments, the composition further includes a biocompatible matrix or scaffold comprising the first composition, the second composition, the third composition, the fourth composition, the fifth composition, or the sixth composition. In some embodiments, the biocompatible matrix or scaffold comprises (i) the adipose progenitor cells and (ii) the first composition, the second composition, the third composition, the fourth composition, the fifth composition, or the sixth composition.

In some embodiments, the method further comprises implanting the matrix or scaffold in a subject in need thereof. In some embodiments, the matrix or scaffold is implanted in or near a soft tissue defect of the subject.

Other objects and features will be in part apparent and in part pointed out hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a series of immunofluorescence images showing location and isolation of CD31 -CD34CD146+/- cells. Adipose tissue are stained with CD34, CD31 and CD146 antibodies, as described in Example 1 . Shown in FIG. 1A-1 H, CD31 -CD34+CD146+ and CD31 -CD34+CD146- are two different populations, with the former mainly marking the pericytes. FIG. 1A is a CD31 stained immunofluorescence image of subcutaneous adipose tissue stained with anti- CD31 antibody showing location and isolation of CD31 -CD34+CD146+ cells. FIG. 1 B is a CD146 stained immunofluorescence image of subcutaneous adipose tissue stained with anti-CD146 antibody showing location and isolation of CD31 -CD34+CD146+ cells. FIG. 1 C is a DAPI stained immunofluorescence image of subcutaneous adipose tissue with nuclei stained with DAPI showing location and isolation of CD31 -CD34+CD146+ cells. FIG. 1 D is a merged image of FIG. 1A, FIG. 1 B, and FIG. 1 C showing location and isolation of CD31 - CD34+CD146+ cells. FIG. 1 E is a CD34 stained immunofluorescence image of subcutaneous adipose tissue stained with anti-CD34 antibody showing location and isolation of CD31 -CD34+CD146- cells. FIG. 1 F is a CD146 stained immunofluorescence image of subcutaneous adipose tissue stained with anti- CD146 antibody showing location and isolation of CD31 -CD34+CD146- cells. FIG. 1 G is a DAPI stained immunofluorescence image of subcutaneous adipose tissue with nuclei stained with DAPI showing location and isolation of CD31 - CD34+CD146- cells. FIG. 1 H is a merged image of FIG. 1 E, FIG. 1 F, and FIG. 1 G showing location and isolation of CD31 -CD34+CD146- cells. Additional details regarding methodology are provided in Example 1 and Example 4. FIG. 2 is a series of scatter plots showing cultured stromal vascular fraction (SVF) cells further sorted by FACS. FIG. 2A is a scatter plot showing cultured stromal vascular fraction (SVF) cells further sorted by FACS using CD31 as a marker. FIG. 2B is a scatter plot showing cultured stromal vascular fraction (SVF) cells further sorted by FACS using CD146 as a marker. Additional details regarding methodology are provided in Example 1 .

FIG. 3 is a series images and histograms showing different

subpopulations of cultured stromal vascular fraction (SVF) cells have different adipogenic capacities. Different subpopulations of the SVF are induced towards adipogenic differentiation, as described in Example 1 . FIG. 3A is an image of SVF with Oil O red staining (for staining of neutral triglycerides, lipids, or lipoproteins). FIG. 3B is an image of CD31 -CD34- cells with Oil O red staining. FIG. 3C is an image of CD31 -CD34+CD146- cells with Oil O red staining. FIG. 3D is an image of CD31 -CD34+CD146+ with Oil O red staining. FIG. 3E is a bar graph showing that the Oil red O staining shows CD31 -CD34+CD146- population displays highest adipogenic capacity. FIG. 3F is a bar graph showing gene expression of PPARy shows CD31 -CD34+CD146- population displays highest adipogenic capacity. FIG. 3G is a bar graph showing gene expression of FABP4 shows CD31 -CD34+CD146- population displays highest adipogenic capacity. FIG. 3H is a bar graph showing HOTAIR is increased during

adipogenesis. FIG. 3I is a bar graph showing CD31 -CD34+CD146- has higher expression of HOTAIR.

FIG. 4 is a pair of images and a pair of histograms showing knockdown of HOTAIR decreased FABP4 expression. FIG. 4A is a bright field image showing SVF cells were transfected with siRNA at high efficiency. FIG. 4B is a

immunofluorescence image showing SVF cells were transfected with siRNA at high efficiency. FIG. 4C is a histogram showing knockdown of HOTAIR can be achieved 7 days after transfection and HOTAIR expression increases (HOTAIR RQ) during adipogenesis at 0, 4, and 7 days for control and siHOTAIR. FIG. 4D shows a histogram showing the adipogenesis marker FABP4 expression

(FABP4 RQ) at 0, 4, and 7 days for control and siHOTAIR, demonstrating knockdown of HOTAIR results in a decreased FABP expression. Additional details regarding methodology are provided in Example 2. FIG. 5 is a series of images and a bar graph showing HOTAIR effects on adipose tissue in C57 mice. FIG. 5A is an image of HE staining of brown, white adipose tissue in C57 mice brown adipose tissue (BAT). FIG. 5B is an image of HE staining of brown, white adipose tissue in C57 mice omental fat. FIG. 5C shows HE staining of adipose tissue in Ob/Ob mice. FIG. 5D is a bar graph showing relative expression of HOTAIR for C57 BAT, C57 omental fat, and Ob/Ob omental fat. Additional details regarding methodology are provided in Example 2.

FIG. 6 is a series of bar graphs showing transfection of PGC1 into adipose stem cells and result on adipogenesis. FIG. 6A shows transfection efficiency of PGC1 a and EGFP into adipose stem cells. FIG. 6B shows UCP1 expression (UCP1 RQ) during overexpression of PGC1 a or EGFP during adipogenesis. FIG. 6C shows PGC1 a expression (PGC1 a RQ) at 0, 4, and 7 days for control and siHOTAIR. FIG. 6D shows UCP1 expression (UCP1 RQ) at 0, 4, and 7 days for control and siHOTAIR. Additional details regarding methodology are provided in Example 2.

FIG. 7 is a plot showing methylation of the UCP1 promoter and effects on adipogenesis. FIG. 7 also shows conservation of the ADRB3 promoter region, as analyzed by the dcode tool. Additional details regarding methodology are provided in Example 3.

FIG. 8 is a plot showing methylation of the UCP1 promoter and effects on adipogenesis. FIG. 8 also shows CpG analysis of human ADRB3 promoter, with the primer for amplification indicated. Additional details regarding methodology are provided in Example 3.

FIG. 9 is a pair of plots showing methylation status of ADRB3 amplicons. FIG. 9A shows representative data of the methylation status of the ADRB3 amplicon 1 (SEQ ID NO: 16). The ADRB3 amplicon 1 had methylated CpG at positions 67, 72, 83, and 160. FIG. 9B shows representative data of the methylation status of the ADRB3 amplicon 2 (SEQ ID NO: 17). The ADRB3 amplicon 2 had methylated CpG at positions 388 and 461 . Methylated CpG are indicated as blue peaks and letters. Additional details regarding methodology are provided in Example 3.

FIG. 10 is a series of plots showing conservation of the UCP1 promoter region. FIG. 10A shows conservation of the UCP1 promoter region, as analyzed by the dcode tool. FIG. 10B shows CpG analysis of human UCP1 promoter, with the primer for amplification indicated. Additional details regarding methodology are provided in Example 3.

FIG. 1 1 is a series of plots and sequences showing methylation status of the UCP1 promoter. FIG. 1 1 A shows representative data of the methylation status of the UCP1 promoter (SEQ ID NO: 14). FIG. 1 1 B shows a schematic illustration of the UCP1 methylation site and its impact on gene expression (SEQ ID NO: 15). Methylated CpG were highlighted in red and the affected transcription binding site were also underlined. Additional details regarding methodology are provided in Example 3.

FIG. 12 is a pair of bar graphs showing relative expression of ADRB3 and UCP1 in response to treatment with a DNA methyltransferase inhibitor. FIG. 12A shows relative expression of ADRB3 in adipogenic stem cells treated with control or 1 μΜ 5-Aza-2'-deoxycytidine ("5-aza-dC") for 7 days, also known as

Decitabine. FIG. 12B shows relative expression of UCP1 in adipogenic stem cells treated with control or 1 μΜ 5-aza-dC for 7 days. Additional details regarding methodology are provided in Example 3. FIG. 13 is a series plots showing exosomes from adipose stem cells. FIG.

13A is a plot of FACS analysis of the cell population of cells isolated from the stromal vascular fraction pO. FIG. 13B is a plot of FACS analysis of the cell population of cells isolated from the stromal vascular fraction p3. Additional details regarding methodology are provided in Example 4. FIG. 14 is a series bar graphs showing different subpopulations of the

SVF induced towards adipogenic differentiation. FIG. 14A shows level of Oil O red staining (for staining of neutral triglycerides, lipids, or lipoproteins) for SVF, CD31 -CD34- cells, CD31 -CD34+CD146- cells, and CD31 -CD34+CD146+ cells. FIG. 14B shows PPARy and adipogenesis marker FABP4 expression for SVF, CD31 -CD34- cells, CD31 -CD34+CD146- cells, and CD31 -CD34+CD146+ cells. FIG. 14C shows relative expression of HOTAIR for CD31 -CD34+CD146- D14 cells and CD31 -CD34+CD146+ D14 cells. Additional details regarding

methodology are provided in Example 4.

FIG. 15 is a series of images and a bar graph showing HUVEC cells were cultured with different conditioned medium (CM). FIG. 15A shows HUVEC cells seeded on gel and cultured with serum free medium for 12h. FIG. 15B shows HUVEC cells cultured with stromal vascular fraction conditioned medium for 12h. FIG. 15C shows HUVEC cells cultured with CD31 -CD34+ conditioned medium for 12h. FIG. 15D shows HUVEC cells cultured with CD31 -CD34+CD146+ conditioned medium for 12h. FIG. 16E shows relative branch length for SF, SVF CM, CD31 -CD34+CM, and CD31 -CD34+CD146+CM. Additional details regarding methodology are provided in Example 4.

FIG. 16 is a diagram and gel image showing exosome isolation. FIG. 16A shows work flow of exosome isolation. FIG. 16B shows a Western Blot for marker, exosome 1 , exosome 2, and exosome 3, with expression of CD63 marked. Additional details regarding methodology are provided in Example 4.

FIG. 17 is a cartoon and series of images showing exosome labeling and endocytosis by endothelial cells. FIG. 17A is a cartoon showing work flow of the exosome labeling and endocytosis by endothelial cells. FIG. 17B shows exosomes with label Cy3 Fluo, bright field, and a merged Image. Additional details regarding methodology are provided in Example 4.

FIG. 18 is a series of bar graphs showing selected miRNAs in adipogenic stem cells. FIG. 18A shows -ACt of miRNAs in SVF cells. U6B expression served as a internal control and the -ACt represents the relative expression of indicated miRNAs. FIG. 18B shows -ACt of miRNAs in exosomes. U6B expression served as a internal control and the -ACt represents the relative expression of indicated miRNAs. FIG. 18C shows relative miRNAs abundance in exosomes. A ration of the -ACt of indicated miRNAs between exosomes and cells above 1 suggests these miRNAs are preferentially secreted into the exosomes. Additional details regarding methodology are provided in Example 4.

FIG. 19 is bar graph showing relative expression of levels of selected miRNAs in CD31 -CD34+CD146- and CD31 -CD34+CD146+ cells. U6B expression served as a internal control. Additional details regarding methodology are provided in Example 4.

FIG. 20 is a series of images and bar graphs showing HOTAIR

expression correlates with white fat adipogenesis and is regulated by

inflammation stimuli. FIG. 20A-20C are images of HE staining of the

interscapular fat, subcutaneous fat, and omental fat from wildtype mice. FIG. 20 20D-20F are images of HE staining of the interscapular fat, subcutaneous fat, and omental fat from Ob/Ob mice. FIG. 20G is a bar graph depicting HOTAIR gene expression in the different adipose depot from both wildtype and Ob/Ob mice. FIG. 20H is an image of p65 (inflammation marker) staining in wildtype subcutaneous fat, which is mainly localized in the cytoplasm. FIG. 20I is an image of p65 (inflammation marker) localized in the nucleus in the subcutaneous fat in ob/ob mice. FIG. 20J is a bar graph showing inflammatory stimuli TNFa could increase HOTAIR expression. FIG. 21 is a series of images showing in situ experiments confirming HOTAIR expression in different adipose tissue. FIG. 21A is an image showing HOTAIR is mainly localized in the nucleus of interscapular tissue. FIG. 21 B is an image showing HOTAIR is mainly localized in the nucleus of inguinal tissue. FIG. 21 C is an image showing is HOTAIR mainly localized in the nucleus of omental tissue.

FIG. 22 is a series of images and bar graphs showing HOTAIR reversely correlates with mitochondria biogenesis and UCP1 expression. FIG. 22A is an image showing mitochondria staining of preadipocytes from brown tissue. FIG. 22B is an image showing mitochondria staining of preadipocytes from

subcutaneous tissue. FIG. 22C is an image showing mitochondria staining of preadipocytes from omental tissue. FIG. 22D is a bar graph showing PGC1 a expression in brown preadipocytes, inguinal preadipocytes, and omental oreadipocytes. FIG. 22E is a bar graph showing UCP1 expression in brown preadipocytes, inguinal preadipocytes, and omental preadipocytes. FIG. 22F is a bar graph showing HOTAIR expression in brown preadipocytes, inguinal preadipocytes, and omental preadipocytes. FIG. 22G and FIG. 22H show that overexpression of PGC1 a in adipocytes (see e.g., FIG. 22G) can increase UCP1 expression (see e.g., FIG. 22H). FIG. 22G is a bar graph showing

overexpression of PGC1 a in adipocytes. FIG. 22H is a bar graph showing increase in UCP1 expression.

FIG. 23 is a series bar graphs showing HOTAIR represses mitochondria biogenesis through PGC1 a. FIG. 23A is a bar graph showing knockdown of HOTAIR increases PGC1 a. FIG. 23B is a bar graph showing knockdown of HOTAIR increases UCP1 .

FIG. 24 is a series of images showing HOTAIR dynamically regulates collagen synthesis in adipogenesis. FIG. 24A is an image of collagen 1/3 staining in brown fat. FIG. 24B is an image of collagen 1/3 staining in inguinal tissue. FIG. 24C is an image of collagen 1/3 staining in omental white fat tissue. FIG. 24D is an image of WT brown fat. FIG. 24E is an image showing knockout of TIMP2, which decreased the extracellular matrix, whitening the brown fat. FIG. 24F is an image showing knockout of TIMP2 increased the volume of omental adipose depot compared to the WT. FIG. 24G is an image of brown fat with a normal diet. FIG. 24H is an image showing a high fat diet also decreased the extracellular matrix and whitened brown fat.

FIG. 25 is a series of data showing overexpression alters the extracellular matrix components by altering the cellular matrix gene expression. The conditioned medium from both EGFP and HOTAIR transfected ASC cells were collected and subjected for mass spectrometry analysis. FIG. 25A is a graph showing go enriched in HOTAIR upregulated genes. FIG. 25B is a graph showing go enriched in HOTAIR downregulated genes.

FIG. 26 is a series of images and graphs. FIG. 26A is a bright field image showing transfection efficiency in fibroblast. FIG. 26B is an immunofluorescence image showing transfection efficiency in fibroblast. FIG. 26C is a bar graph showing overexpression of HOTAIR in fibroblast. FIG. 26D is a bar graph showing overexpression of HOTAIR reduced multiple collagen gene expression. FIG. 26E shows knockdown of HOTAIR increases Col1 a1 expression. FIG. 27 is a series of images showing HOTAIR expression correlates with white fat adipogenesis and regulated by inflammation stimuli. FIG. 27A is an image showing in the wild type mice, collagen is evenly expressed in all the adipocytes. FIG. 27B is an image showing in the wild type mice, collagen is evenly expressed in all the adipocytes. FIG. 27C is an image showing in the wild type mice, collagen is evenly expressed in all the adipocytes. FIG. 27D is an image showing in the wild type mice, collagen is evenly expressed in all the adipocytes. FIG. 27E is an image showing in Ob/Ob mice, CoM a is highly expressed in crown like structure region, suggesting local fibrosis. FIG. 27F is an image showing in Ob/Ob mice, CoM a is highly expressed in crown like structure region, suggesting local fibrosis. FIG. 27G is an image showing in Ob/Ob mice, CoM a is highly expressed in crown like structure region, suggesting local fibrosis. FIG. 27H is an image showing in Ob/Ob mice, CoM a is highly expressed in crown like structure region, suggesting local fibrosis. FIG. 27I is an image showing in Ob/Ob mice, CoM a is highly expressed in crown like structure region, suggesting local fibrosis.

FIG. 28 is an illustration and series of images showing topical injection of AuNP conjugated siRNA can be efficiently target adipose tissue. FIG. 28A is an illustration of an AuNP conjugated siRNA. FIG. 28B is an image of adipose tissue. FIG. 28C is an image of adipose tissue. FIG. 28D is an image of adipose tissue.

FIG. 29 is an illustration of nucleic acid conjugation of AuNPs. The illustration shows AuNPs incubated with thiolated dsDNA or ssRNA, aged with increasing concentrations of NaCI, sonicated, and purified. Additional details regarding methodology are provided in Example 8.

FIG. 30 is a graph showing uptake of 10 nm AuNPs by IL-4 activated ASCs. FIG 30 shows IL-4 stimulated ASCs were found to phagocytose AuNPs more than twice as efficiently. Additional details regarding methodology are provided in Example 8.

FIG. 31 A is a series of graphs showing uptake of 10 nm and 100 nm AuNPs by adipocytes and macrophages. FIG. 31 B is a pie chart representing size preference as a proportion of total observed AuNP phagocytosis in macrophages. FIG. 31 C is a pie chart representing size preference as a proportion of total observed AuNP phagocytosis in adipocytes. Additional details regarding methodology are provided in Example 8.

FIG. 32 is a graph showing uptake of 10 nm AuNPs in brown, inguinal, and omental adipocytes. FIG. 32 demonstrates brown adipocytes were observed to phagocytose most efficiently. Additional details regarding

methodology are provided in Example 8.

FIG. 33A-33D are a series of images showing quantification of

macrophage / adipocyte uptake by immunofluorescence. FIG. 33A is an image of RAW264.7 macrophages. FIG. 33B is an immunofluorescence image of RAW264.7 macrophages. FIG. 33C is an image of adipocytes. FIG. 33C is an immunofluorescence image of adipocytes. Additional details regarding methodology are provided in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based at least in part on the discovery that HOX antisense intergenic RNA "HOTAIR" can induce apidogenesis or modulate formation of fat tissue in vivo. Adipose progenitor cells, such as adipose-derived stem cells (ASCs), can undergo adipogenesis to form new fat. Manipulating key factors involved in adipogenesis, such as HOTAIR, can improve fat grafting protocols.

It is shown herein that overexpression of HOTAIR can result in formation of white adipose tissue. It is also shown herein that inhibition of HOTAIR can result in the formation of brown adipose, which contains an increased amount of blood vessels compared to white adipose tissue. Similar to inhibition of HOTAIR, is shown herein that demethylation of the uncoupler UCP1 promoter can result in transition of white adipose tissue to brown adipose tissue. Further, it is shown herein that exosomes from CD31 -CD34+CD146- stromal vascular fraction cell subpopulation can induce adipogenesis. HOTAIR

As described herein, modulation of HOX antisense intergenic RNA

"HOTAIR" can impact adipogenisis. In some embodiments, expression, overxpression, or administration of HOTAIR can result in formation of white adipose tissue. In some embodiments, knockdown or inhibition of HOTAIR can result in the formation of brown adipose, which contains an increased amount of blood vessels compared to white adipose tissue.

In some embodiments, an agent for modulation of HOTAIR described herein (or a pharmaceutical formulation thereof) can be administered to a subject in need thereof. In some embodiments, an agent increasing expression of HOTAIR described herein (or a pharmaceutical formulation thereof) can be administered to a subject in need thereof. For example, an agent increasing the expression of HOTAIR can be an inflammatory stimuli, such as TNFa (see e.g., FIG. 20). In some embodiments, an agent containing HOTAIR RNA (or a pharmaceutical formulation thereof) can be administered to a subject in need thereof. In some embodiments, an agent knocking down or inhibiting expression of HOTAIR described herein (or a pharmaceutical formulation thereof) can be administered to a subject in need thereof. An agent for modulation of HOTAIR, or HOTAIR RNA itself, can be administered independently or in combination with other compositions described herein, such as an exosome, a miRNA, or a demethylation agent.

Various embodiments described herein employ an adipogenic

composition comprising HOTAIR, or a nucleic acid construct encoding HOTAIR, for promotion of adipogenesis. For example, a controlled release adipogenic composition can be included in a matrix or scaffold so as to promote

differentiation of cells in or on the matrix or scaffold to adipose or adipose-like cells. As another example, a controlled release adipogenic composition can be included in a matrix or scaffold so as to promote adipogenic differentiation of cells that migrated into or onto the matrix or scaffold in response to a cell homing composition also included in the matrix or scaffold. A variety of adipogenic compositions are known in the art (see e.g., Gomillion and Burg 2006

Biomaterials 6052-6063; Poulous et al. 2010 Exp Biol Med 235, 1 185-1 193). As shown herein, HOTAIR can induce or increase apidogenesis in, for example, adipose tissue, adipose derived stem cells, or endothelial cells (see e.g., Example 1 , Example 2). It was also shown that CD31 -CD34+CD146- subpopulation of cells can have increased expression of HOTAIR, where CD31 - CD34+CD146- are shown to have increased adipogenic potential (see e.g., Example 4). It was also shown that HOTAIR expression can increase during adipogenesis (see e.g., Example 2, FIG. 4C). It was also shown that knockdown of HOTAIR can decrease adipogenesis marker FABP4 expression (see e.g., Example 2, FIG. 4D), increased PGC1 a expression (see e.g., Example 2, FIG. 6C), and increased UCP1 expression in adipose tissue (see e.g., Example 2, FIG. 6D).

HOTAIR is a major mammalian long noncoding RNA (IncRNA) that is translated from the HOXC locus. HOTAIR can recognize specific genetic sequences and can recruit the Polycomb chromatin remodeling complex which shuts down gene expression epigenetically through DNA methylation. Studies described herein show the role of HOTAIR in adipogenesis and provide strategies to manipulate cells toward higher adipogenic capacity.

One exemplary HOTAIR gene contains 6,232 bp and encodes 2.2 kb long noncoding RNA molecule, which controls gene expression. The 5' end of HOTAIR is understood to interact with a Polycomb-group protein Polycomb Repressive Complex 2 (PRC2) and as a result regulates chromatin state. It can be required for gene-silencing of the HOXD locus by PRC2. The 3' end of HOTAIR interacts with the histone demethylase LSD1 .

The HOTAIR gene is located within the Homeobox C (HOXC) gene cluster on chromosome 12 and can be co-expressed with HOXC genes. It represses transcription of HOXD genes on chromosome 2 in trans. This gene is shuttled from chromosome 12 to chromosome 2 by Suz-Twelve protein, a component of Polycomb Repressive Complex 2 (PRC2). This gene can interact with both PRC2 and lysine specific demethylase 1 (LSD1 ) complexes through its 5' and 3' domains, respectively, and serves as a scaffold to assemble PRC2 and LSD1 complexes to the HOXD gene cluster. It can couple H3K27 methylation and H3K4 demethylation for epigenetic silencing of HOXD genes in multiple tissues. HOTAIR can also be known as HOXAS, HOXC-AS4, HOXC1 1 -AS1 , or

NCRNA00072.

The HOTAIR gene can be according to NCBI ENSG00000228630, where NCBI ENSG00000228630 refers to a human chromosomal locus, within the Homeobox C (HOXC) gene cluster, comprising 5 IncRNA sequences:

ENST00000424518 = HOTAIR-001 (where genomic sequence = SEQ ID NO: 1 , and cDNA sequence = SEQ ID NO: 2); ENST00000455246 = HOTAIR-002 (where genomic sequence = SEQ ID NO: 3, and cDNA sequence = SEQ ID NO: 4); ENST00000439545 = HOTAIR-003 (where genomic sequence = SEQ ID NO: 5, and cDNA sequence = SEQ ID NO: 6); ENST00000453875 = HOTAIR-004 (where genomic sequence = SEQ ID NO: 7, and cDNA sequence = SEQ ID NO: 8); and ENST00000425595 = HOTAIR-005 (where genomic sequence = SEQ ID NO: 9, and cDNA sequence = SEQ ID NO: 10). HOTAIR-001 spans the entire length of this locus, thus, SEQ ID NO: 1 comprises the entire DNA sequence for the "HOTAIR gene" (ENSG00000228630). The HOTAIR transcript can be according to NCBI ENST00000455246

(genomic sequence is SEQ ID NO: 3, cDNA sequence is SEQ ID NO: 4), ENST00000453875 (genomic sequence is SEQ ID NO: 7, cDNA sequence is SEQ ID NO: 8), ENST00000439545 (genomic sequence = SEQ ID NO: 5, cDNA sequence = SEQ ID NO: 6), ENST00000425595 (genomic sequence is SEQ ID NO: 9, cDNA sequence is SEQ ID NO: 10), or ENST00000424518 (genomic sequence = SEQ ID NO: 1 , cDNA sequence is SEQ ID NO: 2).

HOTAIR nucleic acid sequence can be according to NCBI Accession No. NR_003716 (HOTAIR transcript variant 2 is SEQ ID NO:12), NCBI Accession No. NR_047517(HOTAIR transcript variant 1 is SEQ ID NO:1 1 ), or NCBI Accession No. NR_047518 (HOTAIR transcript variant 3 is SEQ ID NO:13).

HOTAIR can be administered directly as an RNA sequence according to any of the sequences described above, or variants or functional fragments thereof. For example, HOTAIR can be administered in a composition comprising a sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 80% sequence identity thereto (e.g., at least 85%, at least 90%, at least 95%, or at least 99%), or a functional fragment thereof, having an activity associated with HOTAIR.

HOTAIR can be administered in a construct for expression of HOTAIR. A construct contain include a promoter operably linked to a transcribable nucleic acid molecule comprising any of the HOTAIR sequences described above, or variants or functional fragments thereof, operably linked to a 3' transcription termination nucleic acid molecule. For example, a construct for expression of HOTAIR can include a transcribable nucleic acid molecule comprising a sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 80% sequence identity thereto (e.g., at least 85%, at least 90%, at least 95%, or at least 99%), or a functional fragment thereof, having an activity associated with HOTAIR.

Knockdown or inhibition of HOTAIR antisense oligonucleotides can be according to protein aptamers, nucleotide aptamers, or RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), or micro RNAs (miRNA)). Based on target HOTAIR sequences described herein, one of ordinary skill can design and manufacture an agent to knockdown or inhibit HOTAIR. For example, a knockdown or inhibition agent described herein can be specific for a sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 80% sequence identity thereto (e.g., at least 85%, at least 90%, at least 95%, or at least 99%), or a fragment thereof. As another example, a knockdown or inhibition agent described herein can have a sequence comprising SEQ ID NO: 26 (CCGG— aaagcttccacagtgaggact— CTCGAG— agccctcactgtggaagcttt— TTTTTG); SEQ ID NO: 27 (CCGG— aatcagaaaggtcctgctccg— CTCGAG— cggagcaggacctttctatt— TTTTTG); or SEQ ID NO: 28 (CCGG— aaatgtcagagggttctggat— CTCGAG— atccagaaccctctgacattt— TTTTTG), or a sequence having at least 80% sequence identity thereto (e.g., at least 85%, at least 90%, at least 95%, or at least 99%), or a fragment thereof. As another example, an siRNA specific for HOTAIR can be two complimentary RNA sequences. The two complementary strands can be annealed to form a duplex. The complementary strands can be a sense strand and an antisense strand. For example, the sense strand can comprise SEQ ID NO: 29 (GGAGUGAUUAUGCAGUGGGGtt) and the antisense strand can comprise SEQ ID NO: 30 (CCCACUGCAUAAUCACUCCtg), or a sequence having at least 80% sequence identity thereto (e.g., at least 85%, at least 90%, at least 95%, or at least 99%), or a fragment thereof. A stemp-loop siRNA with a linker can also be used. Thus is provided a method to transplant fat by manipulating long noncoding RNA HOTAIR expression in a subject's own adipose-derived stem cells.

TIMP2

As described herein, modulation of TIMP2 can impact apidogenesis. In some embodiments, knockdown of TIMP2 can decrease the extracellular matrix, whitening brown fat. In some embodiments, knockdown of TIMP2 can increase the volume of omental adipose depot.

EXOSOMES

Exosomes derived from adipose tissue (e.g., subpopulation of CD31 - CD34+CD146- adipose stem cells) can be used as a pro-angiogenesis agents (e.g., treatment of ischemic disease or to increase formation of adipose tissue).

In some embodiments, an exosome described herein (or a

pharmaceutical formulation thereof) can be administered to a subject in need thereof. An exosome can be administered independently or in combination with other compositions described herein, such as an agent for modulation of HOTAIR, a miRNA, or a demethylation agent. As shown herein, purified exosomes from adipose stem cells can display abundant expression of CD63, supporting that adipogenic stem cells secrete abundant exosomes robustly (see e.g., Example 4, FIG. 16B). miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25) were identified as important pro-angiogenic components in adipogenic exosomes. Furthermore, it was shown that adipogenic stem cell derived exosomes can efficiently target and enter endothelial cells, thus regulating endothelial migration and

proliferation.

The present disclosure shows that exosomes from CD31 -CD34+CD146- adipose stem cells can be surprisingly strong and effective pro-angiogenic factors. Exosomes are understood to be nano-scale particles that can be secreted by most cell types and function differently due to their origins and target cells. Exosomes have been identified in the conditioned medium of

mesenchymal stem cells and these exosomes have been identified as "trophic factors". But the differences of the exosomes among different subpopulations in mesenchymal stem cells and the detailed effective components in the "trophic factors" are largely unknown. The present disclosure elucidates the pro- angiogenic function of exosomes derived from adipose tissue, stromal vascular fraction (SVF) of adipose tissue, or CD31 -CD34+CD146- subpopulation of adipose stem cells.

An exosome described herein can be derived from adipose tissue. As another example, an exosome can be derived from stromal vascular fraction (SVF) of adipose tissue. For example, an exosome can be derived from mesenchymal stem cells. As another example, an exosome can be derived from adipose stem cells. As another example, an exosome can be derived from stromal vascular fraction (SVF) of adipose tissue. As another example, an exosome can be derived from a CD31 -CD34+CD146- subpopulation of adipose stem cells.

An exosome described herein can have a particle size of about 40 nm to about 120 nm. An exosome described herein can have a particle size of about 80 nm to about 120 nm. An exosome described herein can have an average particle size of about 95 nm to about 105 nm. An exosome described herein can have an average particle size of about 100 nm. As described herein, CD31 -CD34+CD146- adipose stem cell population can display an increased adipogenic capacity or increased expression of

HOTAIR (see e.g., Example 4, FIG. 3, FIG. 14). CD31 -CD34+ conditioned medium can significantly increase branch length of cultured cells (see e.g., Example 4, FIG. 15E). As shown herein, nearly 100% of HUVEC cells can "eat" ASC derived exosomes, supporting that exosomes from ASC can be a viable drug carrier for drug delivery (see e.g., Example 4).

As shown herein, certain miRNAs (e.g., miR126, miR210 (SEQ ID NO: 21 ), miR296 (SEQ ID NO: 22), miR130a (SEQ ID NO: 23)) can be preferentially secreted into the exosomes (see e.g., Example 4, FIG. 18). Levels of miR130a (SEQ ID NO: 23) were shown to be comparable between CD31 -CD34+CD146- and CD31 -CD34+CD146+ subpopulations, while miR221/222 (SEQ ID NO: 24 and SEQ ID NO: 25) were shown to be higher in CD31 -CD34+CD146- cells (see e.g., Example 4, FIG. 19). Thus, miR130a (SEQ ID NO: 23) may have a shared exosome function between the subpopulations. Furthermore, differential expression of miR221/222 (SEQ ID NO: 24 and SEQ ID NO: 25) may explain the increased proangiogenic ability of the CD31 -CD34+CD146- cells. Angiogenic miR221/222 (SEQ ID NO: 24 and SEQ ID NO: 25) in exosome were shown to be the most abundant miRNAs and highly expressed in exosomes from the CD31 - CD34+CD146- subpopulation and have decreased expression in the CD31 - CD34+ CD146+ subpopulation (which has decreased angiogenic capacity).

In some embodiments, an exosome contains one or more of miR126, miR210 (SEQ ID NO: 21 ), miR296 (SEQ ID NO: 22), miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25). In some embodiments, an exosome contains one or more of miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25).

Processes for identification, isolation, or characterization of an exosome are understood in the art (see e.g. Example 4; Jensen 2010 RNA Exosome (Advances in Experimental Medicine and Biology Book 702), Springer,ISBN-10: 1441978402). It is understood in the art that exosomes contain mRNA or microRNA, which can be delivered to another cell, or can be functional in this new location (see e.g., Valadi et al. 2007 Nature Cell biology 9, 654-659). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

Any of the above derived exosomes can be used in compositions or methods described herein.

MIRNA

As described herein, miRNA contained within an adipose tissue-derived exosome can be used to promote angiogenesis. Exosome miRNAs can differ significantly from miRNAs expressed by their parent cells. As such, exosomes can be used to identify miRNA useful for approaches described herein. miRNA associated with pre-angiogenic effects can be useful for approaches described herein.

In some embodiments, one or more miRNA described herein (or a pharmaceutical formulation thereof) can be administered to a subject in need thereof. A miRNA can be administered independently or in combination with other compositions described herein, such as an agent for modulation of HOTAIR, an exosome, or a demethylation agent.

A miRNA associated with exosomes from adipose tissue can be used for a variety of effects associated with the exosome or for independent effects. A miRNA associated with exosomes from adipose tissue can be used to treat a subject for diseases or disorders described herein. Processes for identification or isolation of a miRNA are understood in the art (see e.g., Ochiya 2013 Circulating MicroRNAs: Methods and Protocols (Methods in Molecular Biology), Humana Press, ISBN-10: 1627034528).

Exosome microRNA profiles can be determined according to conventional methods in the art (see e.g., Example 4). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

A miRNA useful in a composition or method described herein can be identified or isolated from an adipose-derived exosome. A miRNA useful in a composition or method described herein can include miR210 (SEQ ID NO: 21 , CUGUGCGUGUGACAGCGGCUGA), miR296 (SEQ ID NO: 22,

agggcccccccucaauccugu), miR130a (SEQ ID NO: 23, cagugcaauguuaaaagggcau), miR221 (SEQ ID NO: 24,

accuggcauacaauguagauuu), or miR222 (SEQ ID NO: 25,

cucaguagccaguguagauccu), or a miRNA having at least about 80% (e.g., at least about 85%, 90%, 95%, or 99%) sequence identity thereto and retaining an activity associated with the miRNA. For example, a miRNA useful in a

composition or method described herein can include miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25), or a miRNA having at least about 80% (e.g., at least about 85%, 90%, 95%, or 99%) sequence identity thereto and retaining an activity associated with the miRNA. As another example, a miRNA useful in a composition or method described herein can include miR221 (SEQ ID NO: 24) or miR222 (SEQ ID NO: 25), or a miRNA having at least about 80% (e.g., at least about 85%, 90%, 95%, or 99%) sequence identity thereto and retaining an activity associated with the miRNA.

A miRNA described herein can be used in a composition or method described herein alone, in combination with one or more other miRNA, RNA, or polypeptides, or in an exosome.

A miRNA described herein can be included in an expression vector, expression construct, plasmid, or recombinant nucleic acid construct. A vector, construct, or plasmid can include a transcribable nucleic acid molecule capable of being transcribed into a miRNA described herein. A transcribable nucleic acid molecule encoding a miRNA described herein can be operably linked to a promoter (e.g., an inducible promoter) functional in vitro or in vivo according to the species of the subject. A transcribable nucleic acid molecule encoding a miRNA described herein can be operably linked to a regulatory sequence. A vector, construct, or plasmid encoding a miRNA described herein can be used to transform a host cell (e.g., in vitro transformation, ex vivo

transformation, in vivo transformation). A host cell transformed with a vector, construct, or plasmid encoding a miRNA described herein can be introduced (e.g., implanted) into a subject according to conventional techniques.

DEMETHYLATION.

A demethylation agent can be used to increase formation of adipose cells tissues. A demethylation agent can be used to increase transition of white adipose tissue to brown adipose tissue. As shown herein, demethylation of the uncoupler UCP1 promoter can promote or induce the transition of white adipose cells or tissue to brown adipose cells or tissue. Observed effects were similar to knockdown of HOTAIR. In some embodiments, a demethylation agent described herein (or a pharmaceutical formulation thereof) can be administered to a subject in need thereof. A demethylation agent can be administered independently or in combination with other compositions described herein, such as an agent for modulation of HOTAIR, an exosome, or miRNA. Uncoupling protein "UCP1 ", also known as Thermogenin is understood to be an uncoupling protein found in the mitochondria of brown adipose tissue. UCP1 can be used to generate heat by non-shivering thermogenesis. UCP1 - mediated heat generation in brown fat can uncouple the respiratory chain, allowing for fast substrate oxidation with a low rate of ATP production. UCP1 is thought to be restricted to brown adipose tissue, where it provides a mechanism for the enormous heat-generating capacity of the tissue.

As described herein, methylated CpG in UCP1 promoter occurred at positions 1645, 1646, 2077, 2078, and 2079. Methylated CpG and the affected transcription binding site are depicted in, for example, FIG. 1 1 B (SEQ ID NO: 15). As shown herein, a known inhibitor of DNA methyltransferase, 5-aza-dC, was used to treat adipogenic stem cell. Inhibition of DNA methyltransferase with 5-aza-dC significantly increased expression of 3-adrenergic receptor (ADRB3) and uncoupling protein 1 (UCP1 ) in adipogenic stem cell (see e.g., FIG. 12A, FIG. 12B). Demethylation agents.

A demethylation agent can be 5-Aza-2'-deoxycytidine ("5-aza-dC"), also known as Decitabine.

5-aza-dC is an epigenetic modifier that inhibits DNA methyltransferase activity, which results in DNA demethylation (hypomethylation) and gene activation by remodeling "opening" chromatin. Genes can be synergistically reactivated when demethylation is combined with histone hyperacetylation.

A demethylation agent can be 5-azacytidine, also known as Azacitidine.

5-azacytidine.

5-azacytidine is a chemical analogue of the cytosine nucleoside used in DNA or RNA. 5-azacytidine is known to inhibit DNA methyltransferase at low doses, causing hypomethylation of DNA.

A demethylation agent can be an antisense oligonucleotide, protein aptamer, nucelotide aptamer, or RNA interference (RNAi) (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNAs (miRNA)). A

demethylation agent can target DNA methyltransferases by degrading their mRNAs and preventing their translation. Antisense RNA, siRNA, shRNA, or miRNA that target DNA methyltransferases are known in the art (see e.g., Yan et al. 2003 Cancer Biol Ther 2(5), 552-556; Schietinger and Reich 2012 Nucleic Acids Research 40(17), 8550-8557; Leu et al. 2003 Cancer Research 63, 61 10). Design of RNA interference specific for DNA methyltransferases is discussed further herein. MATRIX OR SCAFFOLD

One aspect of the present disclosure provides a matrix or scaffold that can be seeded with a composition described herein, for example, adipose progenitor cells or a composition comprising HOTAIR or a nucleic acid construct encoding HOTAIR. The matrix or scaffold including a composition described herein can promote adipogenesis, thereby forming new adipose tissue. In some embodiments, the matrix or scaffold can include living cells, such as adipose progenitor cells. In some embodiments, the matrix or scaffold does not comprise a living cell. Such materials can be used in a procedure to treat or repair a soft tissue defect.

A scaffold can be composed in whole or in part of one or more matrix materials. As used herein, a "matrix" is an amorphous structure, e.g., a gel, in which one or more bioactive ingredients can be suspended. A "scaffold" is understood to have a secondary or tertiary structure (e.g., a columnar structure or a porous structure, such as in a typical collagen sponge, e.g., with fairly uniform pores between about 250 μΜ and 400 μΜ, in which one or more bioactive ingredients can permeate) and may comprise one or more matrix materials. The present disclosure is not limited to any particular matrix or scaffold. Preferably, the matrix or scaffold is biodegradable. In some embodiments, the matrix or scaffold includes a hydrogel. A hydrogel is understood to have a network of polymer chains that can be hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels can be highly absorbent (e.g., over about 80%, 85%, 90%, 95%, 99%, or 99.9% water) natural or synthetic polymers. Hydrogels can also possess a degree of flexibility similar to natural tissue, due to their significant water content. A hydrogel can include, for example, polyvinyl alcohol, sodium polyacrylate, acrylate polymers, or copolymers with hydrophilic groups. Natural hydrogels can include agarose, methylcellulose, hyaluronan, or other naturally derived polymers. A composition described herein can be combined with the matrix or scaffold by any means known in the art. For example, a composition including HOTAIR or a nucleic acid construct encoding HOTAIR can be injected into a matrix or scaffold. As another example, a composition including HOTAIR or a nucleic acid construct encoding HOTAIR can be mixed into a matrix or scaffold. As another example, a composition including HOTAIR or a nucleic acid construct encoding HOTAIR can be encapsulated in the matrix or scaffold, or chemically tethered to, or absorbed in, the matrix or scaffold, by methods known in the art. As another example, adipose progenitor cells can be seeded in the matrix or scaffold.

A matrix or scaffold can be implanted in or near a soft tissue defect. A scaffold described herein can have the shape of a tissue defect of a subject.

A matrix or scaffold can provide a substrate for the growth of cells or formation of tissue. Useful properties of a matrix or scaffold can be porosity, biocompatibility or biodegradability, the ability to support cell growth, or its use as a controlled gene- or protein-delivery vehicle. The three-dimensional

macromolecular structure provided by a scaffold can guide the final shape of bioengineered tissues. Many studies have investigated the role and action of exogenous growth factors in a carrier to deliver the growth factor to an implantation site. Although the carrier may not contribute any additional factors necessary for tissue formation, it can still be an important component of the growth process (Wozney 1990). One of the carrier functions can be to maintain the factor at the site of implantation and thus enhance its local concentration. The carrier also serves as an environment in which tissue can form and therefore helps to define the region in which new tissue can be formed (Whang 1998). Collagenous or synthetic carriers have been used as delivery vehicles, and their physicochemical properties, together with the microenvironment they create, can play a role in the inductive outcome. Carriers can be solid xenogenic {e.g., hydroxyapatite)

(Kuboki 1995, Murata 1998), solid alloplastic (polyethylene polymers) materials (Saito 1998, Isobe 1999), or gels of autogenous (Sweeney 1995, Schwartz 1998), allogenic (Bax 1999, Viljanen 1997), or alloplastic origin (Santos 1998), and combinations of the above (Alpaslan 1996). A matrix or scaffold can further comprise any other bioactive molecule, for example an antibiotic or a chemotactic growth factor. In some embodiments, the matrix or scaffold can be strengthened, through the addition of, e.g., human serum albumin (HSA), hydroxyethyl starch, dextran, or combinations thereof. Suitable concentrations of these compounds for use in the compositions of the application are known to those of skill in the art, or can be readily ascertained without undue experimentation.

The concentration of a composition in a matrix or scaffold will vary with the nature of the composition, its physiological role, or desired therapeutic or diagnostic effect. A therapeutically effective amount can generally be a sufficient concentration of therapeutic agent to display the desired effect without undue toxicity.

A composition can be incorporated into a matrix or scaffold by any known method. In some embodiments, a composition can be imbedded in a gel, e.g., a collagen gel incorporated into the pores of a matrix or scaffold.

Alternatively, chemical modification methods may be used to covalently link a compound on the surface of a matrix or scaffold. The surface functional groups of a matrix or scaffold can be coupled with reactive functional groups of the compound to form covalent bonds using coupling agents well known in the art such as aldehyde compounds, carbodiimides, or the like. Additionally, a spacer molecule can be used to gap the surface reactive groups and the reactive groups of the biomolecules to allow more flexibility of such molecules on the surface of the matrix. Other similar methods of attaching biomolecules to the interior or exterior of a matrix will be known to one of skill in the art.

A composition can alternatively be introduced into or onto the matrix via a carrier based system, such as an encapsulation vehicle. Such vehicles can be useful as slow release compositions. For example, a composition can be microencapsulated to provide for enhanced stability or prolonged delivery.

Encapsulation vehicles include, but are not limited to, microparticles, liposomes, microspheres, or the like, or a combination of any of the above to provide the desired release profile in varying proportions. Other methods of controlled- release delivery of agents will be known to the skilled artisan. Moreover, these or other systems can be combined or modified to optimize the integration/release of agents within the matrix.

Polymeric microspheres can be produced using naturally occurring or synthetic polymers and can be particulate systems in the size range of 0.1 μιτι to 500 μιτι. Polymeric micelles or polymeromes can be polymeric delivery vehicles with similar characteristics to microspheres and can also facilitate encapsulation or matrix integration of a compound described herein. Fabrication,

encapsulation, or stabilization of microspheres for a variety of payloads are within the skill of the art (see e.g., Varde & Pack (2004) Expert Opin. Biol. 4(1 ) 35-51 ). The release rate of the microspheres can be tailored by type of polymer, polymer molecular weight, copolymer composition, excipients added to the microsphere formulation, or microsphere size. Polymer materials useful for forming microspheres include PLA, PLGA, PLGA coated with DPPC, DPPC, DSPC, EVAc, gelatin, albumin, chitosan, dextran, DL-PLG, SDLMs, PEG {e.g., ProMaxx), sodium hyaluronate, diketopiperazine derivatives {e.g.,

Technosphere), calcium phosphate-PEG particles, or oligosaccharide derivative DPPG {e.g., Solidose). Encapsulation can be accomplished, for example, using a water/oil single emulsion method, a water-oil-water double emulsion method, or lyophilization. Several commercial encapsulation technologies are available {e.g., ProLease®, Alkerme).

Liposomes can also be used to integrate a composition with a matrix or scaffold. The agent carrying capacity or release rate of liposomes can depend on the lipid composition, size, charge, drug/lipid ratio, or method of delivery.

Conventional liposomes can be composed of neutral or anionic lipids (natural or synthetic). Commonly used lipids can be lecithins such as phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylserines,

phosphatidylglycerols, or phosphatidylinositols. Liposome encapsulation methods are commonly known in the arts (Galovic et al. (2002) Eur. J. Pharm. Sci. 15, 441 -448; Wagner et al. (2002) J. Liposome Res. 12, 259-270). Targeted liposomes or reactive liposomes can also be used in combination with the agents or matrix. Targeted liposomes have targeting ligands, such as monoclonal antibodies or lectins, attached to their surface, allowing interaction with specific receptors or cell types. Reactive or polymorphic liposomes can include a wide range of liposomes, a common property of liposomes can be their tendency to change their phase or structure upon a particular interaction {e.g., pH-sensitive liposomes). See, e.g., Lasic (1997) Liposomes in Gene Delivery, CRC Press, FL).

A matrix or scaffold can be fabricated with any material recognized as useful by a skilled artisan. Suitable matrix or scaffold materials are discussed in, for example, Ma and Elisseeff, ed. (2005) Scaffolding in Tissue Engineering, CRC, ISBN 1574445219; Saltzman (2004) Tissue Engineering: Engineering Principles for the Design of Replacement Organs and Tissues, Oxford ISBN 019514130X. Non-limiting examples of potentially useful materials for all or part of a matrix or scaffold include poly(ethylene) glycol, poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide), poly(caprolactone), polyanhydride, polyglactin, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates), polyphosphazene, degradable polyurethanes, polyacrylates, ethylene-vinyl acetate polymers or other acyl substituted cellulose acetates or derivatives thereof, polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinyl pyrrolidone, poly(vinylimidazole), chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon®, nylon, agarose, alginate {e.g., calcium alginate gel), fibrin, fibrinogen, fibronectin, collagen {e.g., a collagen gel), gelatin, hyaluronic acid, chitin, or other suitable polymers or biopolymers, or analogs, mixtures, combinations, or derivatives of the above.

In some embodiments, the matrix or scaffold comprises a natural polymer. Exemplary natural polymers can be collagens, chitosan, or polysaccharides. In other embodiments, a matrix or scaffold comprises a synthetic polymer.

Exemplary synthetic polymers can be aliphatic polyesters of poly(a-hydroxy acid)s, or polyethylene glycols. Additional synthetic polymers can be polylactic acid (PLA), polyglycolic acid (PGA), or mixtures of PLA and PGA (PLGA). In some embodiments, the synthetic polymer can be PLGA comprising about 50% PLA and 50% PGA. In other embodiments, a matrix or scaffold comprises a collagen sponge or PLGA.

In some embodiments, a matrix or scaffold has a high porosity. Such a porous structure can provide space for cell migration, adhesion, or the ingrowth of new bone.

Pores or channels of a scaffold can be engineered to be of various diameters. For example, the pores of the scaffold can have a diameter range from micrometers to millimeters. In some embodiments, the pores of the matrix material include microchannels. Microchannels can have an average diameter of about 0.1 μιτι to about 1 ,000 μιτι, e.g., about 50 μιτι to about 500 μιτι (for example about 100 μιτι, 150 μιτι, about 200 μιτι, about 250 μιτι, about 300 μιτι, about 350 μιτι, about 400 μm, about 450 μιτι, about 500 μm, or about 550 μιτι). One skilled in the art will understand that the distribution of microchannel diameters can have any distribution including a normal distribution or a non- normal distribution. In some embodiments, microchannels can be a naturally occurring feature of the matrix material(s). In other embodiments, microchannels can be engineered to occur in the matrix materials.

Several methods can be used for fabrication of porous scaffolds, including particulate leaching, gas foaming, electrospinning, freeze drying, foaming of ceramic from slurry, or the formation of polymeric sponge (Mikos 1994, Mooney 1996, Qing 2002, Sylvain 2006). However, scaffolds prepared using these methods can have some shortcomings in controlling the structure or

interconnectivity of pores, which may limit their application in terms of cell penetration in tissue engineering (Yeong 2004, Tan 2003). A method of making a soft tissue scaffold is additionally provided. The method comprises synthesizing an acellular scaffold in the shape of a tissue defect and adding a compound described herein.

In some embodiments of these methods, a bioactive agent described herein, or nucleic acid encoding such, can be included in the scaffold. Adipose progenitor cells can be present in the matrix at various amounts.

Density-dependent inhibition of cell division (previously termed contact inhibition) can be a factor in cell survival. Too many cells seeded in an engineered tissue or organ scaffold can create shortage of locally available mitogens, growth factors, or survival factors, potentially leading to apoptosis and causing unnecessary waste of in vitro cell expansion time. On the other hand, too few cells seeded in an engineered tissue or organ scaffold can lead to poor regeneration outcome. Various methodologies for optimizing the density of tissue progenitor cells so as to maximize the regenerative outcome of engineered vascularized tissue or organ are known to the art. Various matrix seeding densities can be monitored over time and at end-point cell densities with, for example, histology, structural analysis, immunohistochemistry, biochemical analysis, or mechanical properties. As will be recognized by one skilled in the art, the seeded cell densities of tissue progenitor cells can vary according to, for example, progenitor type, tissue or organ type, matrix material, matrix volume, infusion method, seeding pattern, culture medium, growth factors, incubation time, incubation conditions, or the like. Generally, the adipose progenitor cells can be present in the matrix material at a density of about 0.5 million cells (M) ml^"1 to about 100 M ml^"1. For example, adipose progenitor cells can be present in the matrix material at a density of about 1 M ml^"1, 5 M ml^"1, 10 M ml^"1, 15 M ml^"1, 20 M ml^"1, 25 M ml^"1, 30 M ml^"1, 35 M ml^"1, 40 M ml^"1, 45 M ml^"1, 50 M ml^"1, 55 M ml^"1, 60 M ml^"1, 65 M ml^"1, 70 M ml^" 75 M ml^"1, 80 M ml^"1, 85 M ml^"1, 90 M ml^"1, 95 M ml^"1, or 100 M ml^"1. As another example, the adipose progenitor cells can be present in the matrix material at a density of about 1 M ml^"1 to about 5 M ml^"1 , about 5 M ml^"1 to about 10 M ml^"1 , about 10 M ml^"1 to about 15 M ml^"1, about 15 M ml^"1 to about 20 M ml^"1, about 20 M ml^"1 to about 25 M ml^"1, about 25 M ml^"1 to about 30 M ml^"1, about 30 M ml^"1 to about 35 M ml^"1, about 35 M ml^"1 to about 40 M ml^"1, about 40 M ml^"1 to about 45 M ml^"1, about 45 M ml^"1 to about 50 M ml^"1, about 50 M ml^"1 to about 55 M ml^"1, about 55 M ml^"1 to about 60 M ml^"1, about 60 M ml^"1 to about 65 M ml^"1, about 65 M ml^"1 to about 70 M ml^"1, about 70 M ml^"1 to about 75 M ml^"1, about 75 M ml^"1 to about 80 M ml^"1, about 80 M ml^"1 to about 85 M ml^"1, about 85 M ml^"1 to about 90 M ml^"1, about 90 M ml^"1 to about 95 M ml^"1 , or about 95 M ml^"1 to about 100 M ml^"

1 In some embodiments, progenitor cells used to seed the matrix can be transformed with a heterologous nucleic acid so as to express a bioactive molecule or heterologous protein or to overexpress an endogenous protein. As an example, progenitor cells to be seeded in the matrix can be genetically modified to expresses a fluorescent protein marker. Exemplary markers include GFP, EGFP, BFP, CFP, YFP, or RFP. As another example, progenitor cells to be seeded in the matrix can be genetically modified to express an angiogenesis- related factor, such as activin A, adrenomedullin, aFGF, ALK1 , ALK5, ANF, angiogenin, angiopoietin-1 , angiopoietin-2, angiopoietin-3, angiopoietin-4, angiostatin, angiotropin, angiotensin-2, AtT20-ECGF, betacellulin, bFGF, B61 , bFGF inducing activity, cadherins, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen, collagen receptors αιβι or α₂βι, connexins, Cox-2, ECDGF (endothelial cell-derived growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, endothelins, endostatin, endothelial cell growth inhibitor, endothelial cell-viability maintaining factor, endothelial differentiation shpingolipid G-protein coupled receptor-1 (EDG1 ), ephrins, Epo, HGF, TNF-alpha, TGF-beta, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin or fibronectin receptor α5β1 , Factor X, HB-EGF, HBNF, HGF, HUAF, heart derived inhibitor of vascular cell proliferation, IFN-gamma, IL1 , IGF-2 IFN-gamma, integrin receptors, K-FGF, LIF, leiomyoma-derived growth factor, MCP-1 , macrophage-derived growth factor, monocyte-derived growth factor, MD-ECI, MECIF, MMP 2, MMP3, MMP9, urokiase plasminogen activator, neuropilin (NRP1 , NRP2), neurothelin, nitric oxide donors, nitric oxide synthases (NOSs), notch, occludins, zona occludins, oncostatin M, PDGF, PDGF-B, PDGF receptors, PDGFR-β, PD-ECGF, PAI-2, PD-ECGF, PF4, P1 GF, PKR1 , PKR2, PPAR-gamma, PPAR-gamma ligands, phosphodiesterase, prolactin,

prostacyclin, protein S, smooth muscle cell-derived growth factor, smooth muscle cell-derived migration factor, sphingosine-1 -phosphate-1 (S1 P1 ), Syk, SLP76, tachykinins, TGF-beta, Tie 1 , Tie2, TGF-β, or TGF-β receptors, TIMPs, TNF-alpha, TNF-beta, transferrin, thrombospondin, urokinase, VEGF-A, VEGF- B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF.sub.164, VEGI, EG-VEGF, VEGF receptors, PF4, 16 kDa fragment of prolactin, prostaglandins E1 or E2, steroids, heparin, 1 -butyryl glycerol (monobutyrin), or nicotinic amide. As another example, progenitor cells to be seeded in the matrix can be transfected with genetic sequences that are capable of reducing or eliminating an immune response in the host {e.g., expression of cell surface antigens such as class I or class II histocompatibility antigens can be suppressed). This can allow the transplanted cells to have reduced chance of rejection by the host.

In some embodiments, the matrix material can be seeded with one or more cell types in addition to adipose progenitor cells. Such additional cell type can be selected from thoskin cells, liver cells, heart cells, kidney cells, pancreatic cells, lung cells, bladder cells, stomach cells, intestinal cells, cells of the urogenital tract, breast cells, skeletal muscle cells, skin cells, bone cells, cartilage cells, keratinocytes, hepatocytes, gastro-intestinal cells, epithelial cells, endothelial cells, mammary cells, skeletal muscle cells, smooth muscle cells, parenchymal cells, osteoclasts, or chondrocytes. These cell-types can be introduced prior to, during, or after implantation of the seeded matrix. Such introduction can take place in vitro or in vivo. When the cells are introduced in vivo, the introduction can be at the site of the engineered vascularized tissue or organ composition or at a site removed therefrom. Exemplary routes of administration of the cells include injection or surgical implantation.

ADIPOSE CELLS AND ADIPOSE PROGENITOR CELLS

Various embodiments described herein induce or increase adipogenesis, including formation of adipose or adipose-like cells from progenitor cells.

Adipocytes can be formed from progenitor cells. Adipocytes can be formed from preadipocytes or stem cells, such as mesenchymal stem cells. In various embodiments, an adipose or adipose-like cell can differentiate from a progenitor cell. A progenitor cell can be a cell that is undifferentiated or partially undifferentiated, or can divide or proliferate to produce undifferentiated or partially undifferentiated cells or can differentiate to produce at least one differentiated or specialized cell. A progenitor cell can be a pluripotent cell, which means that the cell is capable of self-renewal or of trans-differentiation into multiple tissue types upon differentiation. Pluripotent progenitor cells include stem cells, such as embryonic stem cells or adult stem cells. A progenitor cell can be a multipotent cell. A progenitor cell can be self-renewing. For example, the progenitor cell can be a stem cell. As another example, the progenitor cell can be an adult stem cell. In some embodiments, a progenitor cell can

differentiate into, or otherwise form, adipocyte cells or adipocyte-like cells. In some embodiments, a progenitor cell can differentiate into, or otherwise form, adipose cells or adipose-like cells. For example, the progenitor cell can be an adipose tissue derived cell, a pre-adipocyte, a mesenchymal stem cell (MSC), an MSC-derived cell, or an adipocyte. Progenitor cells can be isolated, purified, or cultured by a variety of means known to the art. Methods for the isolation or culture of progenitor cells are discussed in, for example, Vunjak-Novakovic and Freshney (2006) Culture of Cells for Tissue Engineering, Wiley-Liss, ISBN-10 0471629359. A progenitor cell can be comprised by, or derived from, an animal, including, but not limited to, mammals, reptiles, or avians, more preferably horses, cows, dogs, cats, sheep, pigs, or chickens, or most preferably human.

In some embodiments, progenitor cells can migrate into a scaffold or matrix material at a density of about 0.0001 million cells (M) ml^"1 to about 1000 M ml^"1. For example, progenitor cells can migrate into a scaffold or matrix material at a density of about 1 M ml^"1, 5 M ml^"1, 10 M ml^"1, 15 M ml^"1, 20 M ml^"1, 25 M ml^" 30 M ml^"1, 35 M ml^"1, 40 M ml^"1, 45 M ml^"1, 50 M ml^"1, 55 M ml^"1, 60 M ml^"1, 65 M ml^"1, 70 M ml^"1, 75 M ml^"1, 80 M ml^"1, 85 M ml^"1, 90 M ml^"1, 95 M ml^"1, or 100 M ml^"1.

Brown adipose tissue or brown fat is understood to be one of two types of fat or adipose tissue (the other being white adipose tissue, or white fat) found in mammals. One function of brown adipose tissue can be to generate body heat (non-shivering thermogenesis) in a subject. Brown adipocytes are known to contain numerous smaller droplets and a much higher number of (iron- containing) mitochondria, which make it brown. Brown fat also contains more capillaries than white fat, since it has a greater need for oxygen than most tissues. Some brown adipose cells have the Myf5 myogenic factor cell surface marker (similar to muscle), which white fat cells do not have. Other brown adipose cells without Myf5 may share the same origin as white fat cells.

Adipocytes can give rise to white fat cells or brown fat cells. Conversion of white adipose cells to brown adipose cells can provide for weight loss in a subject, given that brown fat can take calories from normal fat and burns it. White adipose tissue or white fat is understood to be one of two types of fat or adipose tissue (the other being brown adipose tissue, or brown fat) found in mammals. In a healthy, non-overweight human subject, white adipose tissue composes as much as 20% of the body weight in men and 25% of the body weight in women. White adipose cells contain a single large fat droplet, which forces the nucleus to be squeezed into a thin rim at the periphery.

Adipose (e.g., white adipose or brown adipose) or adipose-like cells, or tissue containing such, can be identified by detecting an adipose-specific marker (see e.g., Poulous et al. 2010 Exp Biol Med 235, 1 185-1 193). For example, adipose or adipose-like cells, or tissue containing such, can be identified by detecting one or more early adipose-specific markers such as ADFP (adipose differentiation related protein, aka adipophilin), pOb24, lipoprotein lipase, or pGH3. As another example, adipose or adipose-like cells, or tissue containing such, can be identified by detecting one or more later adipose-specific markers such as lipogenic enzymes (including glycerophosphate dehydrogenases generally and glycerol-3-phosphate dehydrogenase specifically), aP2, or adipsin. As another example, adipose or adipose-like cells, or tissue containing such, can be identified by detecting adipose stem cells via the CD34 marker. As another example, adipose or adipose-like cells, or tissue containing such, can be identified by detecting accumulation of tri-acyl glycerol. As another example, adipose or adipose-like cells, or tissue containing such, can be identified by detecting lipid accumulation using Oil red-O (see e.g., Example 1 ). An adiposelike cell can be a cell that displays one or more adipose-cell related markers, such as any of those adipose markers described above.

In some embodiments, adipose or adipose-like cells can be formed in a scaffold or matrix material at a density of about 0.0001 million cells (M) ml^"1 to about 1000 M ml^"1. For example, adipose or adipose-like cells can be formed in a scaffold or matrix material at a density of about 1 M ml^"1, 5 M ml^"1, 10 M ml^"1, 15 M ml^"1, 20 M ml^"1, 25 M ml^"1, 30 M ml^"1, 35 M ml^"1, 40 M ml^"1, 45 M ml^"1, 50 M ml^"1, 55 M ml^"1, 60 M ml^"1, 65 M ml^"1, 70 M ml^"1, 75 M ml^"1, 80 M ml^"1, 85 M ml^"1, 90 M ml^"1, 95 M ml^"1, or 100 M ml^"1.

CORE MOLECULE

An agent (e.g., an adipose formation or modulation agent) can include a core molecule coupled to a molecule modulating adipose formation (e.g., siRNA) via a linker group. A core molecule can be, for example, a nanoparticle. A core molecule can include one of more of a gold, or an iron oxide.

As described herein, a core molecule can be about 1 nm to about 1000 nm. For example, a core molecule can be about 1 nm to about 100 nm. As another example, a core molecule can be about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 1 10 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, 700 nm, about 710 nm, about 720 nm, about 730 nm, about 740 nm, about 750 nm, about 760 nm, about 770 nm, about 780 nm, about 790 nm, 800 nm, about 810 nm, about 820 nm, about 830 nm, about 840 nm, about 850 nm, about 860 nm, about 870 nm, about 880 nm, about 890 nm, 900 nm, about 910 nm, about 920 nm, about 930 nm, about 940 nm, about 950 nm, about 960 nm, about 970 nm, about 980 nm, about 990 nm, or 1000 nm.

Gold nanoparticles (AuNPs) can exhibit features such as unusual optical and electronic properties, high stability and biological compatibility, controllable morphology and size dispersion, and easy surface functionalization. AuNPs can be inert, safe, and available in a range of sizes. AuNPs can be produced by reduction of gold salts such as AuCI in an appropriate solvent. A stabilizing agent can also added to prevent the particles from aggregating. Because thiol groups can bind to gold surfaces with high affinity, thiol-modified ligands can be used as stabilizing agents which bind to the surface of the AuNPs by formation of Au- sulfur bonds.

Synthesis of AuNPs with various sizes and shapes can be achieved through judicious choice of experimental conditions and additives.

After synthesis, the stabilizing agents surrounding the AuNPs can be replaced by other molecules by ligand exchange reactions. In addition, ligands can also be linked to the shell of stabilizing agents.

Amino groups can be linked in biological molecules with carboxyl groups at the free ends of the stabilizing agents. Functionalization of AuNPs can make it possible to adjust the surface properties and attach different kinds of molecules to the particles. A core molecule can be an iron oxide. Exemplary iron oxides include superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO). An iron oxide contrast agent can be a commercially available iron oxide. LINKER GROUP

An agent (e.g., an adipose formation or modulation agent) can include a linker group coupling a core molecule and a molecule modulating adipose formation (e.g., siRNA). A linker can be, for example, an organic molecule with at least one end having a functional group. Various linker groups are known in the art. Except as otherwise specified, compositions described herein can include state of the art linker groups. For example, a state of the art linker molecule can be any such molecule capable of coupling a core molecule and molecule modulating adipose formation.

A linker group can include one or more of the following exemplary functional groups: carboxylic acid or carboxylate groups (e.g., Fmoc-protected- 2,3-diaminopropanoic acid, ascorbic acid), silane linkers (e.g.,

aminopropyltrimethoxysilane (APTMS)), or dopamine. Iron on the surface of an iron oxide molecule can be under-coordinated. A linker group, such as carboxylic acid, dopamine, or silane (or another state of the art linker gorup), can provide missing coordination sites (e.g., two oxygen coordination sites) for binding.

A linker group can be any one or more of the following: carboxylic acid or carboxylate groups, Fmoc-protected-2,3-diaminopropanoic acid, ascorbic acid, silane linkers, aminopropyltrimethoxysilane (APTMS), or dopamine.

ACTIVATING FACTOR

An agent (e.g., an adipose formation or modulation agent) can include an activating factor that can increase cellular uptake.

An activating factor can be a cytokine. A cytokine can be a lymphokine, an interleukin, or a chemokine. For example, an activating factor can be an interleukin (IL-4). An activating factor can be a lymphokine, monokine, interferon, colony stimulating factor, or chemokine.

MOLECULAR ENGINEERING

The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid," as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments can be expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

A "promoter" is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product.

Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ISBN-10: 1605500569; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

The "transcription start site" or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1 . With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

"Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably- linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked. A constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3'

transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

"Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, or the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

"Wild-type" refers to a virus or organism found in nature without any known mutation. Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991 ) Gene 97(1 ), 1 19-123;

Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

Nucleotide or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gin by Asn, Val by lie, Leu by lie, and Ser by Thr. Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

"Highly stringent hybridization conditions" are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T_m) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: T_m = 81 .5 °C + 16.6(log-io[Na⁺]) +

0.41 (fraction G/C content) - 0.63(% formamide) - (600/I). Furthermore, the T_m of a DNA:DNA hybrid is decreased by 1 -1 .5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russell, 2006).

Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russell (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ISBN-10: 1605500569; Elhai, J. and Wolk, C. P. 1988. Methods in

Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, or the like. The transfected cells can be selected and

propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term "exogenous" is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of

Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley- VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucelotide aptamers, or RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), or micro RNAs (miRNA)) (see e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, C, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting

deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1 -8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401 -423, describing RNAi). RNAi molecules are commercially available from a variety of sources {e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA

Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs. FORMULATION

The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers. The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of

pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP/NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.

The term "pharmaceutically acceptable excipient," as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for

administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular,

intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in

combination with one or more additional agents or together with other

biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.

Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled- release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired

therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in

temperature, enzymes, water, or other physiological conditions or molecules. Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition. THERAPEUTIC METHODS

Also provided is a process of treating a soft tissue defect in a subject in need administration of a therapeutically effective amount of a composition described herein, so as to modulate expression of HOTAIR, induce or increase adipogenesis, increase formation of white adipose cells or tissue, increase formation of brown adipose cells or tissue, or promote the transition of white adipose cells or tissue to brown adipose cells or tissue. For example, increased expression of HOTAIR in a subject can promote formation of white adipose cells or tissue. As another example, decreased expression of HOTAIR in a subject can promote formation of brown adipose cells or tissue.

Processes for use of exosomes, RNA, or miRNA therapeutically are understood in the art (see e.g., Wood 2014 Exosome Biology and Therapeutics, Wiley-Blackwell, ISBN-10: 1 1 18335805, providing a retrospective review; Jensen 2010 RNA Exosome (Advances in Experimental Medicine and Biology Book 702), Springer,ISBN-10: 1441978402; Sarkar 2014 MicroRNA Targeted Cancer Therapy, Springer, ASIN: B00JVI1WDQ; Lawrie 2013 MicroRNAs in Medicine, Wiley-Blackwell, ASIN: B00H6HIQVU; Guo and Hague 2013 RNA

Nanotechnology and Therapeutics, CRC Press, ASIN: BOODJIVUOO). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing soft tissue defect. A subject in need of the therapeutic methods described herein can be a subject having a need for increase adipogenesis, increase formation of white adipose cells or tissue, increased formation of brown adipose cells or tissue, or increased transition of white adipose cells or tissue to brown adipose cells or tissue.

A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, or humans. For example, the subject can be a human subject.

Generally, a safe and effective amount of a composition described herein is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a composition described herein can increase expression of HOTAIR or induce or increase adipogenesis

According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeutically effective amount of a composition described herein can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to increase

expression of HOTAIR or induce or increase adipogenesis.

The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

As an example, a subject in need can have an adipose (e.g., white, brown, or a combination thereof) cell or tissue deficiency of at least about 5%, about 10%, about 25%, about 50%, about 75%, about 90% or more, or compositions and methods described herein can modulate expression of

HOTAIR, induce or increase adipogenesis, or increase number or function of adipose cells or tissues, increase formation of white adipose cells or tissue, increase formation of brown adipose cells or tissue, or promote the transition of white adipose cells or tissue to brown adipose cells or tissue.

As another example, a subject in need can have damage to a tissue or organ, and the method can provide an increase in biological function of the tissue or organ by at least about 5%, about 10%, about 25%, about 50%, about 75%, about 90%, about 100%, or about 200%, or even by as much as about 300%, about 400%, or about 500%. As yet another example, the subject in need can have an adipose-related disease, disorder, or condition, and the method provides a composition that can induce or increase adipogenesis so as to form adipose cells or tissue sufficient to ameliorate or stabilize the disease, disorder, or condition. For example, the subject can have a disease, disorder, or condition that results in the loss, atrophy, dysfunction, or death of adipose cells. In a further example, the subject in need can have an increased risk of developing a disease, disorder, or condition that is delayed or prevented by the method. As yet another example, the subject in need can have experienced death or dysfunction of adipose cells as the result of a side effect of a medication used for the treatment of another disease or disorder, for example from the use of Copaxone (glatiramer acetate) as a treatment for multiple sclerosis; or from the use of anti-retroviral therapy in HIV-positive individuals. Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or

experimental animals for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.

The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4^th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied

Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired

therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment. Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

Administration of a composition described herein can occur as a single event or over a time course of treatment. For example, a composition described herein can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a soft tissue defect.

A composition described herein can be administered simultaneously or sequentially with another agent, such as an antibiotic, an antiinflammatory, or another agent. For example, a composition described herein can be

administered simultaneously with another agent, such as an antibiotic or an antiinflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a composition described herein, an antibiotic, an antiinflammatory, or another agent.

Simultaneous administration can occur through administration of one

composition containing two or more of a composition described herein, an antibiotic, an antiinflammatory, or another agent. A composition described herein can be administered sequentially with an antibiotic, an antiinflammatory, or another agent. For example, a composition described herein can be

administered before or after administration of an antibiotic, an antiinflammatory, or another agent. ADMINISTRATION

Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or

manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection {e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μηη), nanospheres (e.g., less than 1 μηη), microspheres (e.g., 1 -100 μηη), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in

combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.

Implantation of cells treated with a composition described herein or an engineered construct is within the skill of the art. The cells, scaffold, or matrix material can be either fully or partially implanted into a tissue or organ of the subject to become a functioning part thereof. Preferably, the implant initially attaches to and communicates with the host through a cellular monolayer. Over time, endogenous cells can migrate into the scaffold to form tissue. The cells surrounding the engineered tissue can be attracted by biologically active materials, including biological response modifiers, such as polysaccharides, proteins, peptides, genes, antigens, and antibodies, which can be selectively incorporated into the matrix to provide the needed selectivity, for example, to tether the cell receptors to the matrix, stimulate cell migration into the matrix, or both. The matrix can comprise a gelled phase and interconnecting channels that allow for cell migration, augmented by both biological and physical-chemical gradients. For example, cells surrounding the implanted matrix can be attracted by biologically active materials including IGF1 and bFGF. One of skill in the art will recognize and know how to use other biologically active materials that are appropriate for attracting cells to the matrix.

KITS

Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to a composition described herein, or a matrix, scaffold, or controlled release system for encompassing or encapsulating such. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components. Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.

Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit. Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 )

Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ISBN-10: 1605500569; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747- 754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. The terms "comprise," "have" and "include" are open-ended linking verbs.

Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that "comprises," "has" or "includes" one or more features is not limited to possessing only those one or more features and can cover other unlisted features. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice.

However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

EXAMPLE 1: HOT AIR IN ADIPOGENESIS

The following example explores the role of HOTAIR in adipogenesis.

Subcutaneous WAT were harvested, minced and digested with 1 mg ml^"1 collagenase for 45 min at 37 °C in DMEM/F12 medium containing 1 % BSA and antibiotics. Digested tissues were filtered through sterile 150 μιτι nylon mesh and centrifuged at 250 g for 5 min. The floating fractions consisting of adipocytes were discarded and the pellets representing the stromo-vascular fractions were then resuspended in erythrocyte lysis buffer (154 mM NH₄CI, 10 mM KHCO3, 0.1 mM EDTA) for 10 min to remove red blood cells. The cells were further centrifuged at 500 g for 5 min, plated at 8 10⁵ per well of a 24- well plate and grown at 37 °C in DMEM/F12 supplemented with 10% FBS at 37 °C. ASCs were sorted based on the expression of the surface markers CD146, CD31 , and CD34 by FACS. HOTAIR expression during adipogenesis was verified by RT-qPCR analysis. Cells were transfected with negative control or siHOTAIR, which specifically knocked down the IncRNA HOTAIR. In vitro differentiation was performed using the commercially available Adipogenesis medium (Life Technologies). Adipogenesis was examined by Oil Red O staining or FABP4 expression, a marker for adipocytes.

Results showed that the CD31 -CD34+CD146- subpopulation had higher adipogenic capacity than other adipogenic subpopulations, as shown by Oil Red O staining and FABP4 expression, which is a marker of ad ipocytes(see e.g., FIG. 3). Molecularly, this subpopulation was found to have higher expression of HOTAIR, indicating an association between HOTAIR and adipogenesis.

Preliminary RT-qPCR verified that HOTAIR expression significantly increases over time during adipogenesis. HOTAIR knockdown resulted in decreased FABP4 expression (a marker of adipocytes).

Thus it is shown that HOTAIR plays an integral role in the process of adipogenesis. Results herein further support that HOTAIR manipulation can be used in fat grafting for aesthetics purposes.

Further experiments will show that autologous ASCs with altered HOTAIR expression can more predictably modulate fat grafting in vivo and ultimately be utilized for transplantation in facial reconstruction.

Further experiments will show that HOTAIR is involved in systemic interactions. This supports that targeting HOTAIR expression may provide for anti-obesity therapy.

EXAMPLE 2: HOTAIR EFFECTS ON ADIPOSE TISSUE

The following example studies HOTAIR effects on adipose tissue in C57 mice.

Brown and white adipose tissue was imaged in C57 mice using HE staining (see e.g., FIG. 5A, FIG. 5B). Adipose tissue was imaged in Ob/Ob mice (see e.g., FIG. 5C). And HOTAIR expression was determined among these adipose tissues (see e.g., FIG. 5D).

HOTAIR was transfected into adipose stem cells (see e.g., FIG. 4A, FIG. 4B). Results showed that HOTAIR expression increased during the adipogenesis (see e.g., FIG. 4C). Results also showed that knockdown of HOTAIR decreased adipogenesis marker FABP4 expression (see e.g., FIG. 4D). PGC1 a was transfected into adipose stem cells (see e.g., FIG. 6A).

Results showed that PGC1 a overexpression increased UCP1 expression during adipogenesis (see e.g., FIG. 6B). Results also showed that knockdown of HOTAIR increased PGC1 a expression (see e.g., FIG. 6C) and increased UCP1 expression in adipose tissue (see e.g., FIG. 6D).

EXAMPLE 3: METHYLATION OF UCP1 PROMOTER

The following example studies the effect of methylation of the UCP1 promoter on adipogenesis.

The dcode tool was used to analyze conservation of the ADRB3 promoter region (see e.g., FIG. 7). CpG analysis was performed on human ADRB3 promoter and the primer for amplification (see e.g., FIG. 8).

Results provided representative data of the methylation status of the ADRB3 amplicon 1 (see e.g., FIG. 9A, SEQ ID NO: 16) and the ADRB3 amplicon 2 (see e.g., FIG. 9B, SEQ ID NO: 17). The ADRB3 amplicon 1 had methylated CpG at positions 67, 72, 83, and 160 (see e.g., FIG. 9A, SEQ ID NO: 16). The ADRB3 amplicon 2 had methylated CpG at positions 388 and 461 (see e.g., FIG. 9B, SEQ ID NO: 17).

The dcode tool was used to analyze conservation of the UCP1 promoter region (see e.g., FIG. 10A). CpG analysis was performed on human UCP1 promoter and the primer for amplification (see e.g., FIG. 10B).

Results provided representative data of the methylation status of the UCP1 promoter (see e.g., FIG. 1 1 A) (SEQ ID NO: 14). Results also provided a schematic illustration of the UCP1 methylation site and its impact on gene expression (see e.g., FIG. 1 1 B, SEQ ID NO: 15). Methylated CpG occurred at positions 1645, 1646, 2077, 2078, and 2079 (Methylated CpG highlighted and the affected transcription binding site underlined in FIG. 1 1 B, SEQ ID NO: 15).

A known inhibitor of DNA methyltransferase, 5-Aza-2'-deoxycytidine ("5- aza-dC"), also known as Decitabine, was used to treat adipogenic stem cell.

Adipogenic stem cells were treated with 1 μΜ 5-aza-dc for 7 days and the ADRB3 expression was analyzed by qPCR and GAPDH served an internal control. Results showed significantly increased expression of ADRB3 in adipogenic stem cell treated with 5-aza-dC (see e.g., FIG. 12A).

Adipogenic stem cells were treated with 1 μΜ 5-aza-dc for 7 days and the ADRB3 expression and UCP1 expression was analyzed by qPCR and GAPDH served an internal control. Results showed significantly increased expression of ADRB3 and UCP1 in adipogenic stem cell treated with 5-aza-dC (see e.g., FIG. 12A, FIG. 12B).

EXAMPLE 4: SVF EXOSOMES

The following example shows exosomes from adipose stem cells promote angiogenesis in endothelial cells.

Isolation of adipogenic stem cells. Adipogenic stem/stromal cells were isolated from adipose tissue in the abdomen of subjects. Briefly, adipose tissue was minced with scissors, digested for 30 minutes in DMEM containing 1 mg/ml collagenase type-ll in a shaking water bath at 37°C, and disaggregated through 425-μηη sieves. Mature adipocytes were eliminated by centrifugation at 400 g in ambient temperature for 10 minutes. Cell pellets were resuspended in erythrocyte lysis buffer and incubated for 10 minutes at room temperature, and washed in phosphate- buffered saline. Viable cells were plated on the 75 cm² flask and cultured to confluence. The confluent P0 cells were further sorted based on the surface markers (CD31 , CD34, and CD146) expression. The CD31 -CD34+, CD31 - CD34+CD146-, and CD31 -CD34+CD146+ different subpopulations were used for the following exosome purification, angiogenesis and gene expression analysis.

Purification of the exosomes. The above sorted P0 cells were further plated on the 10 cm dish and cultured for one day. Prior to culture medium collection, ADSCs were washed twice with PBS, and the medium was switched to fresh serum-free medium DMEM. After incubation for 2-3 days, the medium was collected and centrifuged at 2,000 g for 15 min at room temperature and then filtered with a 0.20 μιτι membrane. For exosome purification, the filtered medium was further processed following the manual instruction of the "Total Exosome Isolationt" Kit (Invitrogen, Cat# 4478359).

Labeling of exosomes and endocytosis by endothelial cells.

Purified exosomes derived from SVF (Stromal Vascular Fraction) of adipose tissue were electroporated with Cy3 labeled RNA probe. The labeled exosomes then were incubated with the HUVEC (human umbilical vein endothelial cells) for 6 h and then the cells were washed with PBS twice and observed under fluorescent microscopy.

Angiogenesis assay. A 24-well multiplate stored overnight at -20°C was transferred onto ice and coated with 200 μΙ/well of Geltrex™ LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Invitrogen, Cat# A1413302). 24 h serum starved HUVEC were then seeded at 4x10⁴ cells/well in basal medium and then switch to the conditioned medium or cultured with different exosomes. 12 h after conditioned medium culture or exosome incubation, the angiogenesis results were imaged. miRNA expression analysis.

The purified exosomes and the parental cells were further treated with Trizol for RNA extraction and then miRNA specific reverse-transcription. miScript II RT Kit was applied for miRNA reverse transcription (Qiagen, Cat#218161 ). The relative expression of selected angiogenesis related miRNAs were compared by qPCR using VII-7A ABI realtime machine.

Subcutaneous adipose tissue was isolated and stained with CD34 antibodies (see e.g., FIG. 1 E), CD146 antibodies (see e.g., FIG. 1 F, FIG. 1 B), and CD31 antibodies, and nuclei was stained with DAPI (see e.g., FIG. 1 G, FIG. 1 C). Merged images were obtained (see e.g., FIG. 1 H, FIG. 1 D). Different subpopulations of cells from subcutaneous adipose tissue were identified using CD31 , CD34, and CD146 in FACS analysis (see e.g., FIG. 13A, FIG. 13B).

Different subpopulations of the SVF were induced towards adipogenic differentiation. Results of Oil O red staining (see e.g., FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D) and gene expression (see e.g., FIG. 14A, FIG. 14B, FIG. 14C) show that CD31 -CD34+CD146- population displays highest adipogenic capacity.

Results also showed that CD31 -CD34+CD146- has higher expression of

HOTAIR (see e.g., FIG. 14G).

For an angiogenesis assay, HUVEC cells were cultured with different conditioned medium (CM). HUVEC cells were seeded on the gel and cultured with serum free medium for 12 h (see e.g., FIG. 15A). HUVEC cells were cultured with: stromal vascular fraction conditioned medium for 12 h (see e.g., FIG. 15B); CD31 -CD34+ conditioned medium for 12 h (see e.g., FIG. 15C); and CD31 -CD34+CD146+ conditioned medium for 12 h (see e.g., FIG. 15D). Results showed branch lengths of the above groups (see e.g., FIG. 15E), where there was a significant increase in branch lengths for HUVEC cells cultured with CD31 -CD34+ conditioned medium compared to CD31 -CD34+CD146+

conditioned medium. Work flow of exosome isolation is shown in FIG. 16A. The cell culture medium is collected and centrifuged to delete the cell debris. The resulting supernatant is mixed with the exosome isolation kit at the ratio of 2:1 and then centrifuged at 12,000 g for 1 h. The pellet (comprising exosome) is resuspended in PBS. Exosome identification was performed using exosome markers by Western Blot (see e.g., FIG. 16B). Results showed that the purified exosomes from different samples display abundant expression of CD63, suggesting that adipogenic stem cells secrete abundant exosomes robustly.

Work flow of the exosome labeling and endocytosis by endothelial cells is shown in FIG. 17A. The exosomes isolated from adipogenic stem cells were incorporated with Cy3 RNAi using electroporation and then the exosomes were incubated with HUVEC cells. The labeled exosome can go to the HUVEC cells efficiently (see e.g., FIG. 17B). Results showed that nearly 100% of HUVEC cells can "eat" the ASC derived exosomes, suggesting that exosomes from ASC are a viable drug carrier for drug delivery.

The abundance of selected miRNAs in adipogenic stem cells was determined (see e.g., FIG. 18A). U6B expression served as a internal control and the -ACt in FIG. 18A represents the relative expression of indicated miRNAs. The abundance of selected miRNAs in the exosomes derived from adipogenic stem cells was determined (see e.g., FIG. 18B). U6B expression served as a internal control and the -ACt in FIG. 18B represents the relative expression of indicated miRNAs.

The ratio of the -ACt of indicated miRNAs between exosomes and cells was determined (see e.g., FIG. 18C). A ratio above 1 suggests that these miRNAs (e.g., miR126, miR210 (SEQ ID NO: 21 ), miR296 (SEQ ID NO: 22), miR130a (SEQ ID NO: 23)) are preferentially secreted into the exosomes.

Relative expression of levels of selected miRNAs were determined in CD31 -CD34+CD146- and CD31 -CD34+CD146+ cells (see e.g., FIG. 19). U6B expression served as a internal control. Notably, miR130a (SEQ ID NO: 23) are comparable between CD31 -CD34+CD146- and CD31 -CD34+CD146+

subpopulations, while miR221/222 (SEQ ID NO: 24 and SEQ ID NO: 25) are higher in CD146- cells (see e.g., FIG. 19). miR130a (SEQ ID NO: 23) may have a shared exosome function between the subpopulations. Furthermore, differential expression of miR221/222 (SEQ ID NO: 24 and SEQ ID NO: 25) may explain the compromised proangiogenic ability of the CD31 -CD34+CD146+ cells. EXAMPLE 5: HOTAIR WHITENING BROWN FAT

The following example shows HOTAIR whitens brown fat by targeting both mitochondria and extracellular matrix synthesis.

Results showed HOTAIR expression correlates with white fat

adipogenesis and regulated by inflammation stimuli, TNFa (see e.g., FIG. 20A- 20J). The inflammation marker, p65, was localized in nucleus. Thus, it is thought that inflammation can upregulate HOTAIR expressed in white adipose.

In situ experiments confirmed HOTAIR expression in different adipose tissue. Further, HOTAIR was shown to be mainly localized in the nucleus (see e.g., FIG. 21A-21 B). It was also shown that HOTAIR reversely correlates with mitochondria biogenesis and UCP1 expression (see e.g., FIG. 22A-22H). Thus,

overexpression of PCG1 in adipocytes can increase UCP1 expression.

HOTAIR was shown to repress mitochondria biogenesis through PGC1 a (see e.g., FIG. 4A-4D, FIG. 23A-23B). Experiments showed HOTAIR

dynamically regulates collagen synthesis in adipogenesis (see e.g., FIG. 24A- 24H). It was shown that knockout of TIMP2, decreased the extracellular matrix, and whitened the brown fat (see e.g., FIG. 24D-24E). Thus, modulation of the mitochondria biogenesis and/or modulation of the collagen matrix can modulate whitening of brown fat. The conditioned medium from both EGFP and HOTAIR transfected ASC cells were collected and subjected for mass spectrometry analysis. The results showed overexpression alters the extracellular matrix components by altering the cellular matrix gene expression (see e.g., FIG. 25).

EXAMPLE 6: HOTAIR AND FIBROSIS

The following example shows that HOTAIR overexpresses in transfected fibroblast (a type of cell that synthesizes the extracellular matrix and collagen). It was shown that overexpression of HOTAIR reduced multiple collagen gene expression and knockdown of HOTAIR increased Col1 a1 (e.g., collagen) expression (see e.g., FIG. 26A-26E). Fibrosis was observed in the crown like structure region in obesity. In the wild type mice, collagen was observed to be evenly expressed in all the adipocytes, while in Ob/Ob mice, CoM a is highly expressed in crown like structure region, suggesting local fibrosis (see e.g., FIG. 27A-27H).

EXAMPLE 7: HOTAIR KNOCKDOWN

The following example describes the methods for gene expression knockdown. Knockdown was performed by both siRNA and shRNA. The target sequences used were 5'-aaagcttccacagtgaggact-3' (SEQ ID NO: 18), 5'- aatcagaaaggtcctgctccg-3' (SEQ ID NO: 19), and 5'-aaatgtcagagggttctggat-3' (SEQ ID NO: 20). The siRNA duplexes were purchased from Invitrogen or synthesized by

Genepharm. siRNA duplexes used SEQ ID NO: 29

(GGAGUGAUUAUGCAGUGGGGtt) for the sense strand and SEQ ID NO: 30 (CCCACUGCAUAAUCACUCCtg) for the antisense strand.

The shRNA was prepared by cloning the hairpin structure into PLKO.1 and then packaged into lentivirus. shRNA sequences included SEQ ID NO: 26 (CCGG— aaagcttccacagtgaggact— CTCGAG— agccctcactgtggaagcttt—

TTTTTG); SEQ ID NO: 27 (CCGG— aatcagaaaggtcctgctccg— CTCGAG— cggagcaggacctttctatt— TTTTTG); and SEQ ID NO: 28 (CCGG—

aaatgtcagagggttctggat— CTCGAG— atccagaaccctctgacattt— TTTTTG). The knockdown efficiency was observed to be about 50-70%.

EXAMPLE 8: GOLD NANOPARTICLE (AUNP)-CONJUGATED HOTAIR siRNA

The following example determines the efficiency of gold nanoparticle uptake by adipocytes in RNAi-mediated gene therapy without the use of viral vectors that can be immunogenic. The following Example shows the ability of several types of adipose cells to endocytose gold nanoparticles (AuNPs) demonstrating their utility as vehicles in adipocyte-targeted gene therapy (see e.g., FIG. 32). This study targeted a long noncoding RNA implicated in adipocyte differentiation, HOTAIR, through RNAi.

The objective of the below studies were to examine the uptake efficiency of AuNPs of varying size by ASCs and brown, inguinal, or omental adipocytes and determine the ability of AuNPs functioning as a delivery method for RNAi and knock down gene expression of HOTAIR.

Initial studies showed that AuNP-conjugated siRNA can be injected into brown fat, be uptaken by brown fat, or stimulate formation of brown fat (see e.g., FIG. 28A-28D). This study further demonstrated gold nanopartides were endocytosed efficiently by ASCs. It was shown that a topical injection of gold nanoparticle (AuNP)-conjugated siRNA into brown/white fat tissue can efficiently target adipose tissue (see e.g., FIG. 28A-28D).

This Example shows that, in ASCs, both IL-4 stimulation and smaller particle size will increase AuNP uptake (see e.g., FIG. 30, FIG. 32). Further, brown adipocytes phagocytose more efficiently than their inguinal or omental counterparts (see e.g., FIG. 32). RNAi using AuNPs conjugated to anti-HOTAIR result in a significant decrease in the expression of the RNA.

The ability of several types of adipose cells to endocytose gold

nanopartides (AuNPs) was tested in order to determine their utility as a vehicle in adipocyte-targeted gene therapy. To determine clinical applicability, a long noncoding RNA implicated in adipocyte differentiation, HOTAIR, was targeted through RNAi.

Nucleic acid conjugation of AuNPs: 10 nm and 100 nm colloidal AuNPs (Sigma-Aldrich) were incubated with thiolated dsDNA or ssRNA, aged with increasing concentrations of NaCI (up to 0.3 M), sonicated, and purified (see e.g., FIG. 29).

Spectrophotometric quantification of AuNP uptake: ASCs with and without IL-4 activation as well as brown, inguinal, and omental adipocytes were incubated with conjugated AuNPs. Concentration of AuNPs at time t=0 and t=72 hrs was compared via NanoDrop was compared via NanoDrop.

Quantification of AuNP uptake by immunofluorescence: DNA-conjugated AuNP uptake was compared between RAW264.7 macrophage cells and ASCs using Cy3 labeling.

RNAi knockdown of HOTAIR: anti-HOTAIR and nonsense mRNA- conjugated AuNPs were introduced to ASCs. RT-qPCR was then used to assess relative RNA levels of HOTAIR against stable markers.

ASCs with and without IL-4 were exposed to 10 nm AuNPs for 72 hrs. Media at time t=0 and t=72 was compared through spectrophotometric quantification by NanoDrop at 520 nm absorbance (see e.g., FIG. 30). ASCs were found to phagocytose AuNPs more than twice as efficiently (see e.g., FIG. 30).

Uptake of 10 nm and 100 nm AuNPs in both macrophages and

adipocytes was measured using the aforementioned method. While

macrophages were found to show little preference for either size, adipocytes phagocytosed the larger 100 nm AuNPs much more efficiently than 10 nm AuNPs (see e.g., FIG. 31 ). Pie charts represent size preference as a proportion of total observed AuNP phagocytosis (see e.g., FIG. 31 ).

Brown, inguinal, and omental adipocytes were exposed to 10 nm AuNPs for 72 hrs. Media at time t=0 and t=72 was compared through

spectrophotometric quantification by NanoDrop at 520 nm absorbance. Brown adipocytes were observed to phagocytose most efficiently (see e.g., FIG. 32).

Indirect immunofluorescence was used to label DNA-conjugated AuNPs with Cy3 (see e.g., FIG. 33). Uptake of AuNPs was compared between macrophages and adipocytes via confocal microscopy; levels of AuNP

phagocytosis were found to be comparable between these cell types. ASCs stimulated with IL-4 were found to endocytose AuNPs almost twice as efficiently as controls. While little difference in uptake efficiency was detected between 10 nm and 100 nm AuNPs in macrophages, ASCs showed marked preference for 100 nm AuNPs. Brown adipocytes were observed to endocytose AuNPs much more readily than their inguinal or omental counterparts. Finally, successful knockdown of HOTAIR was achieved in vitro using anti-HOTAIR conjugated AuNPs.

This study demonstrated gold nanoparticles are endocytosed efficiently by ASCs, at levels similar to macrophages, with increased (100 nm) particle size being more selective for ASCs. Application of these findings in the RNAi experiment which successfully knocked down HOTAIR suggests that an adipose-targeted AuNP approach can be a viable therapeutic option in vivo. REFERENCES

Gupta RA, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010 Apr 15;464(7291 ): 1071 -6.

Kato H, et al. Degeneration, regeneration, and cicatrization after fat grafting: dynamic total tissue remodeling during the first 3 months. Plast Reconstr Surg. 2014 Mar;133(3):303e-13e.

Marra KG, and Rubin JP. The potential of adipose-derived stem cells in craniofacial repair and regeneration. Birth Defects Res C Embryo Today. 2012 Mar;96(1 ):95-7.

Claims

Claim 1 . A method of forming adipose tissue in a subject in need thereof, comprising:

contacting an adipose tissue or an adipose progenitor cell of a subject and

(a) a first composition comprising HOX antisense intergenic RNA ("HOTAIR") in an amount effective to increase formation of white adipose tissue or cells;

(b) a second composition comprising a nucleic acid construct encoding HOTAIR, wherein the nucleic acid construct transforms the adipose progenitor cell so as to increase expression of HOTAIR in an amount effective to increase formation of white adipose tissue or cells;

(c) a third composition comprising an RNA interference molecule specific for HOTAIR, wherein the RNA interference molecule decreases expression of HOTAIR by an amount effective to increase formation of brown adipose tissue or cells;

(d) a fourth composition comprising an adipose-derived exosome in an amount effective to increase formation of white or brown adipose tissue or cells;

(e) a fifth composition comprising one or more miRNA selected from the group consisting of miR210 (SEQ ID NO: 21 ), miR296 (SEQ ID NO: 22), miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25), or a miRNA having at least about 95% sequence identity thereto and retaining an activity associated with the miRNA, in an amount effective to increase formation of white or brown adipose tissue or cells; or

(f) a sixth composition comprising a demethylation agent in an amount effective to increase formation of brown adipose tissue or cells.

Claim 2. A composition comprising an adipose formation or modulation agent comprising: (a) a first composition comprising HOX antisense intergenic RNA

("HOTAIR") in an amount effective to increase formation of white adipose tissue or cells;

(b) a second composition comprising a nucleic acid construct encoding HOTAIR, wherein the nucleic acid construct transforms the adipose progenitor cell so as to increase expression of HOTAIR in an amount effective to increase formation of white adipose tissue or cells;

(c) a third composition comprising a protein aptamer, nucleotide aptamer, or RNA interference molecule specific for HOTAIR, wherein the third composition decreases expression of HOTAIR by an amount effective to increase formation of brown adipose tissue or cells;

(d) a fourth composition comprising an adipose-derived exosome in an amount effective to increase formation of white or brown adipose tissue or cells;

(e) a fifth composition comprising one or more miRNA selected from the group consisting of miR210 (SEQ ID NO: 21 ), miR296 (SEQ ID NO: 22), miR130a (SEQ ID NO: 23), miR221 (SEQ ID NO: 24), or miR222 (SEQ ID NO: 25), or a miRNA having at least about 95% sequence identity thereto and retaining an activity associated with the miRNA, in an amount effective to increase formation of white or brown adipose tissue or cells; or

(f) a sixth composition comprising a demethylation agent in an amount effective to increase formation of brown adipose tissue or cells.

Claim 3. The method or composition of any one of claims 1 -2, further comprising a nanoparticle operably linked to (a), (b), (c), (e), or (f).

Claim 4. The method or composition of claim 3, further comprising a cytokine, optionally an interleukin or interleukin-4 (IL-4).

Claim 5. The method or composition of any one of claims 1 -4, wherein first composition comprises SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 95% sequence identity thereto, or a functional fragment thereof, having an activity associated with HOTAIR.

Claim 6. The method or composition of any one of claims 1 -5, wherein second composition comprises an expression construct having a promoter operably linked to a transcribable nucleic acid molecule comprising SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 95% sequence identity thereto, or a functional fragment thereof, having an activity associated with HOTAIR.

Claim 7. The method or composition of any one of claims 1 -6, wherein third composition comprises a protein aptamer, nucleotide aptamer, or RNA interference molecule specific for SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence having at least 95% sequence identity thereto, or a fragment thereof.

Claim 8. The method or composition of any one of claims 1 -7, wherein third composition comprises a protein aptamer, nucleotide aptamer, or RNA interference molecule specific for SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

Claim 9. The method or composition of any one of claims 7-8, wherein the RNA interference molecule comprises a small interfering RNA (siRNA), short hairpin RNA (shRNA), or micro RNA (miRNA).

Claim 10. The method or composition of any one of claims 1 -9, wherein the sixth composition comprises a demethylation agent selected from thr group consisting of (i) 5-Aza-2'-deoxycytidine, (ii) 5-azacytidine, or (iii) an antisense oligonucleotide, protein aptamer, nucelotide aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), or micro RNA (miRNA) specific for a target DNA methyltransferase.

Claim 1 1 . The method of any one of claims 1 or 4-10, wherein contacting adipose tissue or an adipose progenitor cell occurs in vivo.

Claim 12. The method of any one of claims 1 or 4-10, wherein the adipose progenitor cells comprise a CD31 -CD34+CD146- subpopulation of adipose progenitor cells.

Claim 13. The method of any one of claims 1 or 4-12,

wherein contacting adipose tissue or an adipose progenitor cell occurs ex vivo; and

the method further comprises transplanting the contacted adipose progenitor cells into a subject in need thereof.

Claim 14. The method or composition of any one of claims 1 -13, further comprising a biocompatible matrix or scaffold comprising the first composition, the second composition, the third composition, the fourth composition, the fifth composition, or the sixth composition.

Claim 15. The method or composition of any one of claims 1 -14, further comprising a biocompatible matrix or scaffold comprising (i) the adipose progenitor cells and (ii) the first composition, the second composition, the third composition, the fourth composition, the fifth composition, or the sixth

composition.

Claim 16. The method of any one of claims 12-15, further comprising implanting the matrix or scaffold in a subject in need thereof.

Claim 17. The method claim 16, wherein the matrix or scaffold is implanted in or near a soft tissue defect of the subject.