WO2008141177A1

WO2008141177A1 - Methods and compositions for the treatment of sarcoma

Info

Publication number: WO2008141177A1
Application number: PCT/US2008/063241
Authority: WO
Inventors: Francis Y. Lee
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2007-05-11
Filing date: 2008-05-09
Publication date: 2008-11-20
Anticipated expiration: 2009-11-11

Abstract

The present invention is directed to compositions and methods useful in the treatment of sarcomas, such as chondrosarcomas, that are typically resistant to conventional treatment modalities, such as radiotherapy or chemotherapy. Various aspects of the invention involve increasing the sensitivity of a sarcoma to chemotherapy and/or radiotherapy by exposing it to a transformed stem cell, such as a mesenchymal stem cell, that expresses RNAi molecules specific for one or more anti-apoptotic genes and/or multi-drug resistance genes, and optionally expresses a connexin protein that facilitates transfer of the RNAi molecules to sarcomal cells.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF SARCOMA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application Serial No. 60/917,506, filed May 1 1 , 2007, incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

MATERIAL INCORPORATED-BY-REFERENCE

[0003] 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.

FIELD OF THE INVENTION

[0004] The present invention generally relates to methods and compositions for the treatment of sarcomas resistant to conventional treatment modalities.

BACKGROUND

[0005] Chondrosarcoma is the second most common sarcoma arising in bones. Surgical resection is the first treatment of choice for the majority of chondrosarcomas. However, there are a significant number of extensive chondrosarcomas which cannot be surgically removed adequately with a wide margin. Presently, there are no effective chemotherapeutic or radiotherapy regimens for treatment of chondrosarcomas (see e.g., Lee et al. (1999) Journal of Bone and Joint Surgery Am 81 A, 326-338).

[0006] Tissue homeostasis is attained by a delicate balance among cell proliferation, cell survival and cell death. Apoptosis, or programmed cell death, is a special form of cell death regulated by specific pro- and anti-apoptotic proteins, which occurs in both normal and pathologic conditions. Unlike necrosis, apoptosis is governed by a cascade of molecular events that are coordinated by specific regulators. Pro-apoptotic regulators include Bcl-2 antagonist of cell death (BAD), Bcl-2 associated X protein (BAX) and cytochrome c, which are involved in the activation of caspases, the effector molecules of apoptosis. Conversely, inhibitors of apoptosis, such as B-cell leukemia/lymphoma 2 (Bcl-2), Bcl-2 like 1 (Bcl-xL) and x-linked inhibitors of apoptosis (XIAP), counteract pro-apoptotic regulators. Radiotherapy works by triggering apoptosis in cancer cells. There are several molecular mechanisms by which radiation causes cell death. One main mechanism of radiotherapy is the induction of apoptosis by breaking the DNA molecule. Damaging DNA activates BAX (a pro-apoptotic protein) that releases cytochrome c in the mitochondria. Cytochrome c then activates caspases, which ultimately leads to the demise of cancer cells. Despite these well-established mechanisms, chondrosarcomas are known to be resistant to both radiation and chemotherapy (see Bergh et al. (2001 ) Cancer 91 , 1201-1212; Lee et al. (1999) J Bone Joint Surg Am 81 , 326-338; Bovee et al. (2005) Lancet Oncol 6, 599-607).

[0007] Certain cancer cells escape cell death by activating cell survival pathways that involve the inhibitors of apoptosis. For example, upregulation of the anti-apoptotic Bcl-2 family members (e.g., Bcl-2 and Bcl-xL) in certain tumors has been implicated in their chemo-and radio-resistance (see Condon et al. (2002) lnt J Cancer 100, 472-47510; Gilbert et al. (1996) J Cell Physiol 168, 1 14- 122). Chondrosarcomas and other sarcomas are known to express genes that regulate both cell death and cell survival (see Hameetman et al. (2005) Virchows Arch 446, 430-437). It has been reported that the inhibition of apoptosis can lead to tumorigenesis and resistance to chemotherapy and radiotherapy in carcinomas (see Condon et al. (2002) and Gilbert et al. (1996)).

[0008] Bcl-2, Bcl-xL and XIAP are known to inhibit the mitochondria- mediated apoptotic pathway. Using immunohistochemical analysis of human chondrosarcoma tissues, one study showed Bcl-xL expression in all three grades of chondrosarcoma, where the expression of Bcl-xL is strongest in Grade III chondrosarcomas (see Shen et a. (2005) Biochem Biophys Res Commun 328, 375-382).

[0009] Radiation is known to activate the mitochondria-mediated apoptotic process (see Hosokawa et al. (2005) J Radiat Res (Tokyo), 46, 293- 303). The mitochondria-mediated apoptotic pathway, one of three major mechanisms of apoptosis, requires the release of cytochrome c from the mitochondria. Cytochrome c activates caspases that cleave regulatory and structural molecules, and cell death eventually follows (see Ghobhal et al. (2005) CA Cancer J Clin. 55, 178-194). The anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL are known to play a role in antagonizing the mitochondria- mediated apoptotic pathway prior to cytochrome c release from the mitochondria (see Shen et al. (2005) Biochem Biophys Res Commun. 328, 375-382). An increase in the expression of Bcl-2 or Bcl-xL causes resistance to radiation and chemotherapy in different types of cancers such as leukemia, prostate cancer and lung cancer (see Ghobhal et al. (2005) CA Cancer J Clin. 55, 178-194; Fennell et al. (2001 ) Br J Haematol. 1 12, 706-713; Hill (2002) (Review) Oncol Rep. 9. 1 151-1 156).

[0010] It has been reported that Bcl-2 and Bcl-xL can differentially block chemotherapy-induced cell death (see Simonian et al. (1997) Blood. 90, 1208-1216) and radiation-induced cell death (see Lee et al. (1999) Eur J Cancer 35, 1374-1380). It has also been reported that a bispecific antisense oligonucleotide inhibiting both Bcl-2 and Bcl-xL expression induces apoptosis in lung cancer cells in vitro (see Zangemeister-Wittke et al. (2000) Clin Cancer Res. 6: 2547-2555). And an antisense Bcl-2 has been reportedly used as chemo-sensitizer for cancer therapy and has been investigated in clinical trials (see Kim et al. (2004) Cancer. 101 : 2491-2502).

[0011] XIAP (alternate names: MIHA/ hlLP/BIRC4) is a member of the inhibitors of apoptosis protein (IAP) family, which has been identified as the key post-mitochondrial caspase inhibitor (see Devi (2004) Drug News Perspect. 17, 127-134; Holcik et al. (2001 ) Apoptosis 6, 253-26). Unlike the Bcl-2 family proteins, XIAP inhibits both the mitochondria-dependent and -independent apoptotic pathways (see Ghobrial et al. (2005) CA Cancer J Clin 55, 178-194; Devi (2004) Drug News Perspect. 17, 127-134). The mitochondria-independent pathway is known as the cell death-receptor mediated extrinsic pathway. XIAP also inhibits the mitochondria-mediated apoptotic pathway downstream of cytochrome c release from the mitochondria (see Devi (2004); Holcik et al. (2001 )).

[0012] Failure of chemotherapy can result from two major factors. The first is host related, including poor absorption, rapid metabolism and insufficient drug delivery to tumor site. The second is tumor factor, such as loss of a cell surface receptor or transporter for a drug, specific metabolism of a drug, mutation of the specific target of a drug, and/or the increase in drug efflux (see Gottesman (2002)).

[0013] P-glycoprotein, a product of multi-drug resistance gene-1 (MDR-1 ), and anti-apoptotic protein overexpression are two common mechanisms of chemo-resistance in tumor cells. P-glycoprotein is a transmembrane ATP-dependent pump which exports drugs out of cells as protection against toxins. High expression of P-glycoprotein and anti-apoptotic proteins has been reported in chondrosarcoma cells (see Kim et al. (2007); Rosier et al. (1997); Shen et al. (2005); and Terek et al. (1998)). The major mechanism of the multi-drug resistance is P-glycoprotein expression of tumor cells (see Ueda et al. (1987)). Several in vitro studies have reported that most chondrosarcoma cells express P-glycoprotein to confer MDR (see Rosier et al. (1998) and Wyman et al. (1999)). This MDR resulting from overexpression of P- glycoprotein has been reported in different types of soft tissue sarcomas and hematological malignancies (see Sonneveld (2000) and Stein et al. (1996)). In addition to drug transportation, P-glycoprotein overexpressing cells exhibit abrogation of mitochondrial cytochrome c release and caspase-3 activation, which may be dependent on Bcl-xL overexpression (see Kojima et al. (1998)).

[0014] Doxorubicin is a drug widely used in cancer chemotherapy. It is an anthracycline antibiotic and also intercalates DNA. Doxorubicin has been reported to remain after washout of the doxorubicin treatment in P-glycoprotein expressing chondrosarcoma cells although the amount is less than chondrosarcoma cells which do not express P-glycoprotein (see Wyman et al. (1999)).

[0015] Human mesenchymal stem cells (hMSC) are useful for delivery of gene therapy because of immunosuppressive effect and easy in vitro growth and maintenance (see Ramasamy et al. (2007) Leukemia 21 :304-310). Tumor cells recruit hMSCs to make tissue stroma, as evidenced by hMSCs homing to tumor sites in other studies (see Studney et al. (2004) J. Natl. Cancer Inst. 96. 21 :1593-1603). Further, it has been shown that hMSCs produce connexin 43, necessary to form the gap junctions to allow siRNA transfer between cells (see Valiunas et al. (2006) J. Physiol. 568(Pt 2):459-68).

SUMMARY OF THE INVENTION

[0016] Among the various aspects of the present invention is the provision of a therapeutic approach to increase the sensitivity of sarcomas, especially chondrosarcomas, to conventional treatment modalities, such as chemotherapy and/or radiotherapy. Such an approach is valuable with regard to, for example, chondrosarcoma treatment, which is typically resistant to conventional chemotherapy and/or radiotherapy treatment and cannot always be surgically removed with adequately wide margins.

[0017] Briefly, therefore, the present invention is directed to increasing the sensitivity of resistant sarcomas to conventional treatment protocols by exposing the resistant sarcoma to RNAi molecules specific for anti-apoptotic genes and/or multi-drug resistance genes responsible, at least in part, for the resistance. After such exposure to RNAi molecules, conventional treatment modalities are more effective against the formerly-resistant sarcomas.

[0018] The present teachings include methods for increasing sensitivity of a sarcoma to radiotherapy or chemotherapy by introducing an RNA interference molecule specific for an anti-apoptotic gene and/or a multi-drug resistance gene to sarcoma cells. In one embodiment, the method of increasing sensitivity of a sarcoma to radiotherapy or chemotherapy includes, inter alia, introducing into a subject in need thereof an effective amount of a composition that includes a transformed stem cell with a nucleic acid encoding an RNA interference (RNAi) molecule specific for a messenger RNA (mRNA) corresponding to at least one anti-apoptotic gene and/or a multi-drug resistance gene, wherein the transformed stem cell expresses the RNAi molecule.

[0019] The present teachings also include a method of treating a sarcoma resistant to chemotherapy or radiotherapy with an RNA interference molecule specific for an anti-apoptotic gene and/or a multi-drug resistance gene to sarcoma cells. In one embodiment, the method of treating a sarcoma resistant to chemotherapy or radiotherapy includes, inter alia, introducing into a subject in need thereof an effective amount of a composition that includes a transformed stem cell with a nucleic acid encoding an RNA interference (RNAi) molecule specific for a messenger RNA (mRNA) corresponding to at least one anti-apoptotic gene and/or a multi-drug resistance gene, wherein the transformed stem cell expresses the RNAi molecule; and exposing the subject to chemotherapy or radiotherapy.

[0020] The present teachings also include a composition for treatment of sarcoma that contains a transformed stem cell expressing an anti-apoptotic gene-specific RNA interference (RNAi) molecule and/or a multi-drug resistance gene-specific RNA interference (RNAi) molecule. In one embodiment, the composition for treatment of sarcoma includes, inter alia, a transformed stem cell with a nucleic acid encoding an RNA interference (RNAi) molecule specific for a messenger RNA (mRNA) corresponding to at least one anti-apoptotic gene and/or a multi-drug resistance gene and a pharmaceutically acceptable carrier, wherein the transformed stem cell expresses the RNAi molecule.

[0021] In various embodiments, the sarcoma is a chondrosarcoma resistant to radiotherapy or chemotherapy.

[0022] In various embodiments, the nucleic acid encodes RNAi molecules specific for mRNAs corresponding to at least two genes independently selected from an anti-apoptotic gene and a multi-drug resistance gene.

[0023] In various embodiments, the anti-apoptotic gene(s) is/are selected from the group consisting of Bcl-2, Bcl-xL, and XIAP. In various embodiments, the multi-drug resistance gene is a P-glycoprotein gene. In some configurations, the nucleic acid comprises RNAi molecules specific for mRNAs corresponding to at least one anti-apoptotic gene and at least one multi-drug resistance gene.

[0024] In various embodiments, the RNAi molecule is a double- stranded RNA (dsRNA), small interfering RNA (siRNA), hairpin RNA (shRNA), multicistronic siRNA, or microRNA (miRNA). In some embodiments, the RNAi molecule is a small interfering RNA (siRNA). In various embodiments, the RNAi has a length of about 19 nucleotides to about 25 nucleotides.

[0025] In various embodiments, the stem cell is a mesenchymal stem cell.

[0026] In various embodiments, the transformed stem cell also includes a nucleic acid encoding a connexin protein, wherein the transformed stem cell expresses the connexin protein and the connexin protein facilitates transfer of RNAi molecules from the transformed stem cell to a sarcoma cell. In some embodiments, the nucleic acid has a promoter that is upregulated by expression of the connexin protein. In some embodiments, the connexin protein is a connexin32 protein, connexin37 protein, connexin40 protein, connexin43 protein, or connexin45 protein. In some embodiments, the nucleic acid has an osteocalcin (OC) promoter or a bone sialoprotein (BSP) promoter, wherein the OC promoter or the BSP promoter is upregulated by the connexin protein, and wherein the upregulation of the OC promoter or the BSP promoter results in increased expression levels of RNAi molecules.

[0027] In various embodiments, the subject is a mammal.

[0028] In various embodiments, the amount of introduced composition includes about 1x10⁸ to about 1x10² of the transformed stem cells.

[0029] Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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.

[0031] Figure 1 is a schematic diagram demonstrating a molecular pathway of apoptosis triggered by radiation.

[0032] Figure 2 depicts a histogram and a series of immunoblots. Figure 2A is a histogram depicting detection of apoptosis by flow cytometry after radiation. Figure 2B is a series of immunoblots of anti-apoptotic proteins Bcl-2, Bcl-xL, and XIAP.

[0033] Figure 3 depicts a series of immunoblots and a histogram. Figure 3A contains immunoblots showing decreased expression of Bcl-2, Bcl-xL, and XIAP by siRNA over a 3 day period; where - is carrier only, and NS is non- silencing siRNA. Figure 3B contains immunoblots showing single and dual gene silencing. Combinatorial siRNA treatments (Bcl-2+Bcl-xL, Bcl-2+XIAP or BcI- xL+XIAP siRNA) silenced two target proteins. Quantitative expression of each protein was plotted in the histogram.

[0034] Figure 4 depicts a series of microscopic images and a series of histograms. Figure 4A contains a microscope picture after siRNA and radiation treatment; original magnification x100. Figure 4B contains a series of histograms depicting the effect of gene silencing on radio-sensitivity in chondrosarcoma cells; where NS is non-silencing siRNA.

[0035] Figure 5 depicts a series of images of colony formation and a pair of histograms. Figure 5A contains images of cell culture plates showing survival and proliferation of chondrosarcoma cells after gene silencing. Dark- grey dots indicate colonies positively stained with crystal violet. Figure 5B is a pair of histograms showing clonogenic survival after radiation and siRNA.

[0036] Figure 6 is a histogram showing apoptosis after siRNA treatment. The apoptosis rate is provided for two cell lines, SW1353 and JJ012, with RNAi silencing of Bcl-2, Bcl-xLm, and XIAP in different combinations.

[0037] Figure 7 is a schematic diagram demonstrating the regulation of apoptosis by P-glycoprotein and cell survival genes in response to chemotherapy.

[0038] Figure 8 depicts a histogram and a series of immunoblots. Figure 8A is a histogram depicting detection of apoptosis by flow cytometry after chemotherapy. Two normal articular chondrocytes (NC1 and NC2), two chondrosarcoma cells (SW1353 and JJ012), and human embryonic kidney cells (HEK) were tested. Figure 8B is a series of immunoblots of the multi-drug resistance gene product P-glycoprotein, as well as anti-apoptotic proteins Bcl-2, Bcl-xL, and XIAP.

[0039] Figure 9 depicts a series of plots generated by flow cytometric analysis. Figure 9A shows cell surface P-glycoprotein expression following antibody staining using IgG as an isotype control and an anti-P-glycoprotein antibody, PG. Figure 9B shows doxorubicin uptake is increased as doxorubicin concentration increases in two chondrosarcoma cell lines. Figure 9C shows doxorubicin levels in cells decreases in a time dependent manner.

[0040] Figure 10 depicts a fluorescent micrograph of transfected cells and a series of immunoblots. Figure 10A shows fluorescent (light-grey) cytoplasm in chondrosarcoma cells 24 hours after transfection with fluorescence- tagged siRNA. Figure 1 OB shows target protein expression of each siRNA is significantly decreased in comparison to control groups.

[0041] Figure 1 1 depicts a series of light micrographs and a pair of histograms. Figure 1 1A original magnification x100. Figure 1 1 B shows the effect of gene silencing on chemo-sensitivity in chondrosarcoma cells quantitatively; where NS is non-silencing siRNA, and PG is P-glycoprotein.

[0042] Figure 12 depicts a pair of histograms showing P-glycoprotein and anti-apoptotic gene silencing with chemotherapy and the effect on cell survival and proliferation.

[0043] Figure 13 depicts a schematic representation of targeted siRNA delivery via hMSC.

[0044] Figure 14 depicts a series of microscopic images showing 1 :1 ratio of hMSC:osteosarcoma cells at 24 hours post co-culture. Figure 14A is a black-and-white light micrograph showing a synctium of osteosarcoma cells. Figure 14B is a fluorescent image showing the location of siRNA fluorescence (light-grey dots). Figure 14C is a fluorescent image showing GFP-tagged osteosarcoma cells (light-grey shapes; e.g. lower-left quadrant). Figure 14D is a composite of images from figures A, B and C.

[0045] Figure 15 depicts a series of microscopic images showing 7:1 ratio hMSC:osteosarcoma cells 24 hours post-co-culture. Figure 15A is a black- and-white light micrograph showing a hMSC adjacent to a GFP-tagged osteosarcoma cell in the upper-left quadrant. Figure 15B is a fluorescent image showing a GFP-tagged osteosarcoma cell (light-grey) in the upper left quadrant. The red fluorescence (light-grey) in figure 15C reveals siRNA transfected hMSCs. Figure 15D is a composite of images from figures of 15B and C showing that the GFP-tagged osteosarcoma cell in the upper-left quadrant is transfected with siRNA via the adjacent hMSC. [0046] Figure 16 depicts a schematic representation of an in vivo method of osteosarcoma treatment using siRNA and stem cells.

DETAILED DESCRIPTION OF THE INVENTION

[0047] Applicants have discovered that chondrosarcoma cells overexpress Bcl-2 (see e.g. SEQ ID NO: 1 ; NCBI Accession no. NM 000633 or NM_000657), Bcl-xL (see e.g. SEQ ID NO: 2; NCBI Accession no. NM 138578 or NM_001 191 ), and XIAP (see e.g. SEQ ID NO: 3; NCBI Accession no. NM 001 167), all of which can promote cell survival after radiation and/or chemotherapy treatment. Applicants have also discovered that chondrosarcoma cells overexpress P-glycoprotein (see e.g. SEQ ID NO: 4; NCBI Accession no. NM_000927), which can promote cell survival following chemotherapy. While the following mechanistic understanding does not in any way limit the invention, it is thought that chondrosarcoma cells may escape radiation-induced, or chemical-induced, apoptosis by increasing the expression of anti-apoptotic proteins such as Bcl-2 (see e.g. SEQ ID NO: 5; NCBI Accession no. NP_000624), Bcl-xL (see e.g. SEQ ID NO: 6; NCBI Accession no. NP_612815), XIAP (see e.g. SEQ ID NO: 7; NCBI Accession no. NP 001 158), and/or multidrug resistance proteins such as P-glycoprotein (see e.g. SEQ ID NO: 8; NCBI Accession no. NP 000918). As such, anti-apoptotic and/or multi-drug resistance gene silencing can enhance cancer treatment modalities, such as radiotherapy and chemotherapy, in sarcoma cells typically resistant to such treatment modalities by facilitating apoptotic pathways.

[0048] Silencing of anti-apoptotic genes can have a therapeutic role as a molecular adjuvant treatment of sarcomas, such as chondrosarcoma, when combined with other modalities of treatment, such as radiation treatment, chemotherapy treatment, and/or surgical resection. Furthermore, silencing of multiple anti-apoptotic genes can further enhance the efficacy of other treatment modalities. For example, double gene silencing can further enhance radio- sensitivity (see e.g., Example 1 ). Preferably, there is a synergistic effect of multiple anti-apoptotic gene silencing on apoptosis of tumor cells in conjunction with other treatment modalities. For example, silencing of Bcl-xL and XIAP can synergistically enhance radio-sensitivity by eight-fold or more.

[0049] Silencing of multi-drug resistance genes can have a therapeutic role as a molecular adjuvant treatment of sarcomas, such as chondrosarcoma, when combined with other modalities of treatment, such as radiation treatment, chemotherapy treatment, and/or surgical resection. Furthermore, silencing of multiple multi-drug resistance genes can further enhance the efficacy of other treatment modalities. For example, double gene silencing can further enhance chemo-sensitivity (see Example 4). Preferably, there is a synergistic effect of multiple multi-drug resistance gene silencing on apoptosis of tumor cells in conjunction with other treatment modalities.

[0050] Various embodiments of the methods described herein involve introduction of RNAi molecules specific for one or more anti-apoptotic genes into sarcoma cells or tissues containing sarcoma cells, where the sarcoma cells are at least in part resistant to conventional treatment modalities. Such methods can increase the sensitivity of sarcoma cells, such as chondrosarcoma cells, to conventional treatment modalities, such as radiotherapy or chemotherapy. Such methods generally include contacting a composition containing RNAi molecules specific for one or more anti-apoptotic genes with chondrosarcoma cells, for example via RNAi loaded progenitor cells, preferably via RNAi loaded stem cells, and more preferably RNAi loaded stem cells expressing or overexpressing a connexin protein.

[0051] Various embodiments of the methods described herein involve introduction of RNAi molecules specific for one or more multi-drug resistance genes into sarcoma cells or tissues containing sarcoma cells, where the sarcoma cells are at least in part resistant to conventional treatment modalities. Such methods can increase the sensitivity of sarcoma cells, such as chondrosarcoma cells, to conventional treatment modalities, such as radiotherapy or chemotherapy. Such methods generally include contacting a composition containing RNAi molecules specific for one or more multi-drug resistance genes with chondrosarcoma cells, for example via RNAi loaded progenitor cells, preferably via RNAi loaded stem cells, and more preferably RNAi loaded stem cells expressing or overexpressing a connexin protein.

[0052] In accordance with one method, a composition containing RNAi loaded tissue progenitor cells is introduced into tissue containing chondrosarcoma cells in a subject. The tissue progenitor cell can be, for example, a mesenchymal stem cell (MSC), MSC-dehved cell, osteoblast, chondrocyte, myocyte, adipocyte, neuronal cell, neuronal supporting cells such as Schwann cells, neural glial cells, fibroblastic cells including interstitial fibroblasts, tendon fibroblasts or tenocytes, ligament fibroblasts, periodontal fibroblasts, craniofacial fibroblasts, gingival fibroblasts, periodontal fibroblasts, cardiomyocytes, epithelial cells, dermal fibroblasts, liver cells, uretheral cells, kidney cells, periosteal cells, bladder cells, or beta-pancreatic islet cell. Tissue progenitor cells, preferably stem cells and more preferably mesenchymal stem cells, can be isolated by a variety of means known to the art. For the balance of the discussion, reference will be made to stem cells and/or mesenchymal stem cells. Such further discussion is understood to also apply, or be adaptable, to tissue progenitor cells generally.

[0053] In brief, the method of introducing a composition containing RNAi loaded tissue progenitor cells into tissue containing chondrosarcoma cells in a subject method can be performed as follows. Once isolated, the stem cells can be purified and/or expanded. In some embodiments, the isolated stem cells can then be transformed with a nucleic acid that encodes RNAi molecules specific for one or more anti-apoptotic genes, such that the RNAi molecules are expressed by the transformed stem cell. In some embodiments, the isolated stem cells can then be transformed with a nucleic acid that encodes RNAi molecules specific for one or more multi-drug resistance genes, such that the RNAi molecules are expressed by the transformed stem cell. In further embodiments, the isolated stem cells can then be transformed with a nucleic acid that encodes RNAi molecules specific for one or more anti-apoptotic genes and one or more multi-drug resistance genes, such that the RNAi molecules are expressed by the transformed stem cell.

[0054] The isolated stem cells can also be transformed with a nucleic acid that encodes a connexin protein. Such transformed stem cell can express or overexpress a connexin, such as connexin43, to facilitate transfer of RNAi molecules from the transformed stem cell to chondrosarcoma cells. RNAi loaded stem cells can then be formulated as a composition comprising the transformed stem cells along with, for example, a pharmaceutically acceptable carrier or adjuvant.

[0055] The composition so formed can then be introduced into tissue of the subject that contains a sarcoma, such as chondrosarcoma, or alternatively, systemically introduced. The subject will usually have been diagnosed as having, or being at risk for, a sarcoma. For example, the subject can have been diagnosed as having, or being at risk for, chondrosarcoma.

[0056] Introduction of the composition can be according to methods generally known to the art. For example, a RNAi loaded transformed stem cell composition can be administered to a subject by way of direct injection into the chondrosarcoma. Introduction of RNAi loaded stem cells can be a single occurrence or can occur sequentially over a period of time selected by the attending physician. The time course and number of occurrences of transformed stem cell introduction into a subject can be dictated by monitoring the chondrosarcoma in conjunction with other treatment modalities, where such methods of assessment and devisement of treatment course are within the skill of the art of an attending physician.

[0057] It should be recognized that methods of this invention can readily be practiced in conjunction with existing cancer therapies to effectively treat or prevent sarcomas (such as chondrosarcoma), where such existing cancer therapies were not necessarily effective against such sarcoma prior to the present approach. ANTI-APOPTOTIC GENES

[0058] In various embodiments, RNAi molecules are used to degrade homologous cellular mRNAs that specifically encode one or more anti-apoptotic proteins in sarcoma cells (e.g., chondrosarcoma cells). Preferably, at least two anti-apoptotic genes are silenced. Target anti-apoptotic genes include pre- mitochondrial apoptotic inhibitors (e.g., Bcl-2; Bcl-xL) or post-mitochondhal apoptotic inhibitors (e.g., XIAP) (see e.g., Figure 1 ). Such combinatorial approaches can incapacitate cell survival machinery more effectively than silencing a single anti-apoptotic gene. Examples of anti-apoptotic genes that can be silenced according to methods described herein include, but are not limited to, Bcl-2; Bcl-xL (i.e., Bcl-2 like 1 ); XIAP (x-linked inhibitors of apoptosis; i.e., BIRC4, ILP1 , or MIHA); BIRC8; CARD8; CFLAR; FADD; SERPINB9, and survivin.

[0059] Preferably, anti-apoptotic genes that can be silenced according to methods described herein are Bcl-2; Bcl-xl; and XIAP (see e.g., Example 1 ; infra for discussion regarding RNAi gene silencing). For example, Bcl-2 and Bcl- xl can be silenced. As another example, Bcl-2 and XIAP can be silenced. As another example, Bcl-xL and XIAP can be silenced. As another example, Bcl-2, Bcl-xl, and XIAP can be silenced.

MULTI-DRUG RESISTANCE GENES

[0060] In various embodiments, RNAi molecules are used to degrade homologous cellular mRNAs that specifically encode one or more multi-drug resistance proteins in sarcoma cells, such as chondrosarcoma cells. Target multi-drug resistance genes include transmembrane pumps, such as P- glycoprotein. For example, RNAi molecules can be used to degrade homologous cellular mRNAs that specifically encode P-glycoprotein, a multi-drug resistance protein, in chondrosarcoma cells (see e.g., Figure 7).

[0061] Preferably, multi-drug resistance genes that can be silenced according to methods described herein are P-glycoprotein (see e.g., Example 4). Combinatorial approaches can incapacitate cell survival machinery more effectively than silencing a single multi-drug resistance gene and/or anti- apoptotic gene. A multi-drug resistance gene (e.g., P-glycoprotein) can be silenced in conjunction with silencing of one or more anti-apoptotic genes. As an example, P-glycoprotein can be silenced in combination with XIAP, Bcl-2, and/or Bcl-xl.

SARCOMAS

[0062] The methods described herein can be directed to various sarcomas that are resistant to conventional treatment modalities. Preferably, the sarcoma is a chondrosarcoma. The balance of the discussion will be directed to chondrosarcoma, but one skilled in the art will recognize that the disclosure below also applies to other sarcomas and can be adapted accordingly.

[0063] The aggressiveness of chondrosarcoma is usually graded based on how fast it grows and its likelihood to metastasize or spread to other parts of the body. Generally, Grade 1 is a low grade (slow growing) cancer, and grades 2 and 3 (or even 4) are higher grades (fast growing) cancers (see generally, Longe, ed. (2005) The Gale Encyclopedia Of Cancer, 2nd edition, Thomson Gale, 1419 p., ISBN-10: 1414403623; Harsh et al., ed. (2003) Chordomas and Chondrosarcomas of the Skull Base and Spine, Thieme Medical Publishers, 372 p., ISBN-10: 0865779856). The methods described herein can be directed to chondrosarcomas diagnosed as corresponding to various grades, such as Grade I chondrosarcomas, Grade Il chondrosarcomas, Grade III chondrosarcomas, or Grade IV chondrosarcomas. For example, methods described herein can be directed to Grade Il chondrosarcomas, given their high resistance to both chemo- and radiotherapy, retention of a cartilage phenotype, metastatic potential, and high recurrence rate (see e.g., Example 1 ).

[0064] The methods described herein can be directed to various bones of the body containing a chondrosarcoma or formerly containing a chondrosarcoma (e.g., after surgical rescission). For example, the methods described herein can be directed to the ribs, sternum, pelvic bone, spine, shoulder bones, superior regions of the arms and legs, and/or the skull, or other bones. Preferably, the methods described herein are directed to chondrosarcomas in areas which cannot be surgically removed adequately with a wide margin, such as the pelvis, spine, or skull.

RNAi

[0065] Various embodiments employ RNAi molecules to degrade homologous cellular mRNAs that specifically encode anti-apoptotic proteins in sarcoma (e.g., chondrosarcoma) cells, thereby decreasing the availability of such factors and ultimately increasing the susceptibility of chondrosarcoma cells to various treatment modalities, such as radiotherapy or chemotherapy. RNAi molecules include, but are not limited to double-stranded RNA (dsRNA), small interfering RNA (siRNA), hairpin RNAs (shRNAs), multicistronic siRNA, and microRNA (miRNA). RNAi in mammalian somatic cells generally requires identification of individual small interfering RNAs (siRNAs). Preferably, the RNAi molecules for use in the methods described herein are siRNA. siRNAs against specified sequences are commercially available or can be synthesized using known oligonucleotide synthetic techniques.

[0066] RNA interference is the process by which an RNAi molecule, for example an siRNA, specifically suppresses the expression of a gene bearing its complementary sequence. Post-transcriptional gene silencing that is induced by RNAi molecules is achieved through RNA-RNA sequence recognition and base pairing. Suppression of an anti-apoptotic gene inhibits the production of the corresponding anti-apoptotic protein. RNAi is well understood in the art. Generally, exogenously provided synthetic siRNAs are converted into active functional siRNAs by an endogenous kinase that provides 5'-phosphate groups in the presence of ATP. Then, siRNAs assemble into endoribonuclease- containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA. Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand.

[0067] The levels of anti-apoptotic proteins in chondrosarcoma cells can be down-regulated by RNA interference by administering to the patient a therapeutically effective amount of siRNAs, or other RNAi molecules, specific for these targets. siRNA specific for anti-apoptotic gene products such as Bcl-2 (see e.g. Ambion catalogue no. 42815), Bcl-xL (see e.g. Ambion catalogue no. 6876), or XIAP (see e.g., Ambion catalogue no. 2553), and multi-drug resistance genes such as P-glycoprotein (see e.g. Ambion catalogue nos. s10419, s10418, s10420, 1 18137, 4123, and/or 3933), are available from commercial sources (e.g., Ambion, Austin, TX). Examples of siRNA for use in the methods described herein include, but are not limited to, Bcl-2 siRNA (SEQ ID NO: 9), Bcl-xL siRNA (SEQ ID NO: 10), and XIAP siRNA (SEQ ID NO: 1 1 ). The RNAi molecules can be administered to the subject by any means suitable for delivering the RNAi molecules to the cells of the tissue at, in, or near the chondrosarcoma. For example, siRNA can be administered by gene gun, electroporation, lipophilic agents, or by other suitable parenteral or enteral administration routes, such as intravitreous injection. Preferably, the RNAi molecules specific for anti-apoptotic gene products are transfected into a stem cell and the stem cell then administered to the subject. More preferebly, siRNA specific for anti-apoptotic gene products is co-transfected into a stem cell (e.g., a mesenchymal stem cell) along with a gene for expressing a connexin protein or a connexin promoter (e.g., a connexin43 or a connexin43 promoter).

[0068] The siRNA specific for an anti-apoptotic gene will usually comprises short double-stranded RNA that can be from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length, and more preferably about 21 to about 23 nucleotides in length, that are complementary to a mRNA sequence corresponding to an anti-apoptotic gene. [0069] The si RNA can be targeted to any stretch of approximately 19- 25 contiguous nucleotides in any of the mRNA target sequences. Target sequences can be selected from, for example: the mRNA sequence of P- glycoprotein; Bcl-2; Bcl-xL; and/or XIAP. The anti-apoptotic target sequences are preferably mammalian, and more preferably human. Searches of the human genome database (BLAST) can be carried out to ensure that selected siRNA sequence will not target other gene transcripts. Techniques for selecting target sequences for siRNA are known in the art (see e.g., Elbashir et al. (2001 ) Nature 41 1 , 494-498; Mittal (2004) Nature Reviews, Genetics 5, 355-365). Thus, the sense strand of the present siRNA comprises a nucleotide sequence identical, or substantially identical, to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA of an anti-apoptotic gene. Generally, a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3' direction) from the start codon. The target sequence can, however, be located in the 5' or 3' untranslated regions, or in the region nearby the start codon.

[0070] An effective amount of siRNA can be an amount sufficient to cause RNAi-mediated degradation of the target anti-apoptotic mRNA, or, in conjunction with an additional treatment modality, an amount sufficient to sensitize the chondrosarcoma to the treatment modality and thereby provide for inhibition of the progression of chondrosarcoma in a subject. One skilled in the art can readily determine an effective amount of the siRNA of the invention to be administered to a given subject by taking into account factors such as the size and weight of the subject; the type of chondrosarcoma ; the extent of the chondrosarcoma penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of siRNA comprises an intercellular concentration at or near the chondrosarcoma site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 1 O nM. It is contemplated that greater or lesser amounts of siRNA can be administered.

[0071] The instant invention uses siRNA, which functions to bind to and degrade RNA in the living cell causing the down-regulation of a specific gene. Methods to down-regulate a target gene via the use of siRNA are well known and well established in the art. Exemplary methods that cause degradation of RNA in a mammalian that may function in accord with methods described herein include, but are not limited to, those described in U.S. Patent Application Numbers 09/821 ,832, 10/490,955, 10/255,568, 10/832,248, 10/433,050, 10/832,432, 10/832,257, 11/142,865, 1 1/142,866, 1 1/474,738, 1 1/474,919, 1 1/474,930, 1 1/474,932, 10/349,320, 10/384,260, 10/382,634, 10/382,768, 10/382,395, 09/866,557, 10/055,797, 09/858,862, 10/350,798, 10/997,086, 1 1/330,043, 10/759,841 , 10/646,070, 10/821 ,710, 1 1/218,999, 1 1/180,928 , 10/821 ,726, 10/346,853, 09/997,905, 09/100,813, 09/646,807, 09/100,812, 90/007,247, 90/008,096, 09/215,257, 10/283,267, 10/283,190 and 10/282,996 (each of which are incorporated herein by reference).

PLATFORM

[0072] Anti-apoptotic and/or multi-drug resistance gene silencing agents, for example RNAi molecules, can be transfected into cells ex vivo, with the transfected cells subsequently targeted to a sarcoma, such as a chondrosarcoma, of a subject. Preferably the transfected cell is a stem cell, and more preferably, the stem cell is a mesenchymal stem cell. Systemically infused mesenchymal stem cells may have the ability to home to sites of active tumohgenesis (see e.g., Studeny et al. (2004) J. Natl. Cancer Inst. 96, 1593- 1603; Nakamura et al. (2004) Gene Ther. 1 1 , 1 155-1 164). As such, both tissue targeted delivery as well as systemic delivery of RNAi loaded mesenchymal stem cells is contemplated. The balance of discussion will be directed to engineered stem cells, but it is understood that the following can be adapted to cells of other types and/or various stages of differentiation. [0073] Stem cells can be isolated, purified, cultured, and differentiated by a variety of means known to the art (see e.g., Challen and Little (2006) Stem Cells 24(1 ), 3-12; Lanza et al., eds. (2004) Handbook of Stem Cells, Academic Press, ISBN 0124366430; Lanza et al., eds. (2005) Essentials of Stem Cell Biology, Academic Press, ISBN 0120884429; Saltzman (2004) Tissue Engineering: Engineering Principles for the Design of Replacement Organs and Tissues, Oxford ISBN 01951413OX; Vunjak-Novakovic and Freshney, eds. (2006) Culture of Cells for Tissue Engineering, Wiley-Liss, ISBN 0471629359; Minuth et al. (2005) Tissue Engineering: From Cell Biology to Artificial Organs, John Wiley & Sons, ISBN 352731 1866). As will be appreciated by one skilled in the art, the time between isolation, culture, expansion, and/or implantation can vary according to particular application. Incubation (and subsequent replication and/or differentiation) of the engineered composition containing stem cells can be, for example, at least in part in vitro, substantially in vitro, at least in part in vivo, or substantially in vivo. Determination of optimal culture time is within the skill of the art.

[0074] The stem cells can be derived from the same or different species as the transplant recipient. For example, the stem cells can be derived from an animal, including, but not limited to, mammals, reptiles, and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human. It is also contemplated that autologous stem cells can be obtained from the subject, into which the stem cells are reintroduced after transformation to express RNAi molecules. Such autologous stem cells can be expanded and/or transformed, as described herein, before re-introduction to the host.

[0075] Stem cells can be obtained by screening a plurality of cells from donors. The population of cells to be screened are, preferably, those of bone marrow. But stem cells can be obtained from any tissue known to contain such cells (see generally Challen and Little (2006) Stem Cells 24(1 ), 3-12). After screening, stem cells can be selected and prepared for transformation and subsequent introduction into the subject.

[0076] If desired, the stem cells can be expanded ex vivo (or in vitro) using, for example, standard methods used to culture stem cells and maintain stable cell lines. Alternatively, these cells can be expanded in vivo {i.e., after implantation). These cells can also be used for future transplantation procedures. The group of screened and isolated cells can, optionally, be further enriched for stem cells prior to introduction into the subject. Methods to select for stem cells are well known in the art (e.g., MoFlow Cell Sorter). For example, samples can be enriched by tagging cell-surface markers of undifferentiated stem cells with fluorescently labeled monoclonal antibodies and sorting via fluorescence-activated cell sorting (FACS).

[0077] The RNAi molecules specific for one or more anti-apoptotic genes and/or multi-drug resistance genes can be transfected into stem cells (e.g., mesenchymal stem cells), so as to be stably expressed, by a variety of ways known to the art. Molecular transfection processes are well known (see e.g. Sambrook and Russel (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN 0879697717; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN 0879695773; Ausubel et al. (2002) Short Protocols in Molecular Biology, Current Protocols, ISBN 0471250929; Spector et al. (1998) Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN 0879695226). Except as otherwise noted herein, therefore, the engineering of a stem cell to overexpress an anti-apoptotic gene silencing agent and/or a multi-drug resistance gene silencing agent, such as RNAi molecules specific for one or more anti-apoptotic and/or a multi-drug resistance genes, can be carried out in accordance with such processes.

[0078] As an example, siRNA expression cassettes can be integrated into a plasmid and the plasmid then transfected into a stem cell. Such plasmids are inexpensive to produce, often available commercially, and can provide continuous expression of hairpin RNAs (shRNAs). The shRNAs contain a double stranded stem of about 19 to about 29 base pairs identical in sequence to the target mRNA; the two strands are usually connected by a loop of about 6 to about 9 bases. The loop is removed in vivo by the Dicer enzyme to generate effective siRNAs. Various promoters can be used to obtain stable and efficient expression of the RNAi molecules. Examples of such promoters include, but are not limited to, RNA polymerase III (pol III) promoters, tRNA promoters, and RNA- pol-ll-based cytomegalovirus (CMV) promoters. Preferably, the promoter is a pol III promoter, which is generally active in all cell types and efficiently directs the synthesis of small, non-coding transcripts that bear well-defined ends. Other methods of transforming stem cells to express RNAi molecules specific for one or more anti-apoptotic genes will be known to one skilled in the art.

[0079] In various embodiments, the engineered cell, for example an engineered stem cell, expresses (or overexpresses) a connexin. Connexins are gap junction proteins that form aqueous intercellular channels, which can allow the diffusion of small molecules and ions from cell to cell (e.g., RNAi molecules from an engineered mesenchymal stem cell to a chondrosarcoma cell). Generally, each gap junction pore is formed by juxtaposition of two hemichannels in adjacent cells, where each hemichannel is composed of a hexamehc array of transmembrane connexin proteins (see e.g., Plotnikov et al. (2003) Circulation 108, IV-547; and Valiunaset al. (2002) , Cir. Res. 91 , 104- 1 1 1 ). Examples of connexins include, but are not limited to, Cx23, Cx25, Cx26, Cx30.2, Cx30, Cx31.9, Cx30.3, Cx31 , Cx32, Cx36, Cx37, Cx40.1 , Cx40, Cx43, Cx45, Cx46, Cx47, Cx50, Cx59, and Cx62. Preferably, the connexin expressed in the transformed cell is Cx32, Cx37, Cx40, Cx43, or Cx45. Transformation of a stem cell (e.g., a mesenchymal stem cell) to stably express or overexpress a connexin can be performed according to methods known in the art (see e.g., Example 2). Isolated stem cells (e.g., RNAi loaded mesenchymal stem cells) can be transduced with, for example, a lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector, or other vector system, overexpressing a connexin gene. Expression or overexpression of a connexin protein can increase gap junctional permeability (see e.g., Example 2). Increased gap junction permeability can allow transfer of expressed RNAi molecules described herein to cells of target tissues. Expression or overexpression of a connexin protein can also increase expression of genes linked to osteoblast specific promoters, such as the osteocalcin (OC) promoter or the bone sialoprotein (BSP) promoter (see e.g., Example 2).

[0080] In transformed stem cells that express or overexpress a connexin protein, expression of an RNAi molecule can be increased by coupling to a promoter which is upregulated by the connexin protein. In various embodiments, a nucleotide encoding an RNAi molecule specific for an anti- apoptotic and/or a multi-drug resistance gene can be coupled to a promoter which is upregulated by a connexin protein. For example, the RNAi encoding nucleotide can be coupled to an osteocalcin (OC) promoter or a bone sialoprotein (BSP) promoter, both of which are shown to be upregulated in cells expressing or overexpressing connexin43 protein (see e.g., Example 2).

[0081] Preferably, the stem cell stably expresses connexin43, and more preferably, the stem cell stably overexpresses connexin43. Connexin43 is a 43kDa gap junction protein with GenBank accession number BC026329 and RefSeq ID NM_000165. Expression, or overexpression, of connexin43 can facilitate the transfer of RNAi molecules expressed in the transformed stem cell into, around, or near target sarcoma cells, such as chondrosarcoma cells. As discussed above, connexin43 can facilitate transfer of RNAi molecules specific for one or more anti-apoptotic genes from the mesenchymal stem cell into a target chondrosarcoma cell and/or upregulate expression of RNAi molecules linked to promoters such as the OC promoter or the BSP promoter.

[0082] Preferred embodiments for delivery of RNAi via gap junctions of engineered cells are as described in: WO/20050591 1 1 , "Delivery of DNA or RNA via gap junctions from host cells to target cells and a cell-based delivery system for antisense or siRNA"; WO/2004065580, "Mesenchymal stem cells as a vehicle for ion channel transfer in syncytial structures"; US 2004/0137621 , "Mesenchymal stem cells as a vehicle for ion channel transfer in syncytial structures"; US 2005/00029_.1_.4 , "Mesenchymal stem cells as a vehicle for ion channel transfer in syncytial structures"; Plotnikov et al. (2003) Circulation 108, IV-547; and Valiunaset al. (2002) , Cir. Res. 91 , 104-1 1 1 ; each of which are incorporated herein by reference in their entirety.

[0083] Stem cells transformed to express RNAi molecules and/or a connexin can optionally be further transformed with a heterologous nucleic acid so as to express one or more additional bioactive molecules or heterologous proteins or to overexpress an endogenous or exogenous protein, or variants thereof. As an example, RNAi loaded stem cells can be genetically modified to expresses a fluorescent protein marker (e.g., GFP, EGFP, BFP, CFP, YFP, RFP). Marker protein expression can be especially useful in implantation scenarios, as described herein, so as to monitor stem cell placement, retention, and replication in target tissue. As another example, RNAi loaded stem cells 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 and class Il histocompatibility antigens may be suppressed). This can allow the transplanted cells to have reduced chance of rejection by the host, especially where the cells were from a different subject. A protein is understood to include a protein, protein domain, polypeptide, or peptide, and any fragment or variant thereof having protein function. A protein variant has similar biological activity and at least 60% sequence identity (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 99.9%) to the protein of interest.

[0084] As used herein, "sequence identity" means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. Sequence identity is present when a subunit position in both of the two sequences is occupied by the same nucleotide or amino acid, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

[0085] In various embodiments, the stem cells transfected with anti- apoptotic gene silencing agents and/or a multi-drug resistance gene silencing agents can be labeled so as to allow tracking of the distribution of such cells within the subject over time. For example, the mesenchymal stem cells transfected to overexpress RNAi molecules specific for anti-apoptotic genes and/or multi-drug resistance genes can be labeled with magnetic particles (e.g., superparamagnetic iron oxide particles), which allows for in vivo tracking of the cells by magnetic resonance imaging (MRI) by producing a characteristic hypointense area resulting in a signal void within the MRI image. As another example, mesenchymal stem cells transfected to overexpress RNAi molecules specific for anti-apoptotic genes and/or multi-drug resistance genes can be labeled with a conjugated fluorophore to allow for identification in histopathological sections.

[0086] The amount of RNAi loaded stem cells introduced into a subject can be that amount sufficient to cause RNAi-mediated degradation of the target anti-apoptotic and/or multi-drug resistance mRNA in the target tissue, or, in conjunction with another treatment modality, an amount sufficient to sensitize the sarcoma (e.g., chondrosarcoma) to the treatment modality and thereby provide for inhibition of the progression of sarcoma in a subject. Sensitization of the sarcoma may be readily assessed and determined by the skilled artisan, based on known procedures. One skilled in the art can readily determine an effective number of RNAi-loaded stem cells to be administered to a given subject by taking into account factors such as the size and weight of the subject; the extent of the sarcoma penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. An effective amount of cells can be, for example, about 1 x10⁸ to about 100 cells. For example, about 1x10⁸, about 1 x10⁷, about 1x10⁶, about 1 x10⁵, about 1x10⁴, about 1 x10³, or about 1 x10² cells. Preferably, about 1 x10⁶ to about 1 x10⁵ cells are introduced. It is contemplated that greater or lesser amounts of RNAi loaded stem cells can be administered.

[0087] Introduction of RNAi loaded stem cells into a subject can occur before, during, or after one or more sarcoma treatment modalities, such as surgical resection, radiotherapy, and/or chemotherapy. Introduction of RNAi loaded stem cells into a subject can be performed in conjunction with alternate modes of therapeutic treatment especially where a chondrosarcoma cannot be surgically removed adequately with a wide margin or where the chondrosarcoma is recurrent.

NEED FOR TREATMENT

[0088] A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the sarcoma (e.g., chondrosarcoma) at issue. For example, the diagnosis of chondrosarcoma can serve to identify a subject with a need for a therapy described herein. The subject is preferably an animal, including, but not limited to, mammals, reptiles, and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human. Diagnosis of sarcoma, including chondrosarcoma, is within the skill of the art (see generally, Longe, ed. (2005) The Gale Encyclopedia Of Cancer, 2nd edition, Thomson Gale, 1419 p., ISBN- 10: 1414403623; Harsh et al., ed. (2003) Chordomas and Chondrosarcomas of the Skull Base and Spine, Thieme Medical Publishers, 372 p., ISBN-10: 0865779856). Subjects with an identified need of therapy include those with diagnosed chondrosarcoma or an indication of chondrosarcoma amenable to therapeutic treatment described herein and subjects who have been treated, are being treated, or will be treated for a chondrosarcoma.

[0089] Introduction of a composition comprising an RNAi molecule specific for one or more anti-apoptotic genes and/or multi-drug resistance genes can occur before, during, or after one or more sarcoma treatment modalities, such as surgical resection, radiotherapy, and/or chemotherapy (see generally, Longe, ed. (2005) The Gale Encyclopedia Of Cancer, 2nd edition, Thomson Gale, 1419 p., ISBN-10: 1414403623; Harsh et al., ed. (2003) Chordomas and Chondrosarcomas of the Skull Base and Spine, Thieme Medical Publishers, 372 p., ISBN-10: 0865779856; Doherty (2005) Current Surgical Diagnosis & Treatment, 12th ed., McGraw-Hill Medical, 1490 p., ISBN-10: 007142315X). Introduction of RNAi molecules into a subject can be performed in conjunction with alternate modes of therapeutic treatment, especially where a sarcoma such as a chondrosarcoma cannot be surgically removed adequately with a wide margin or where chondrosarcoma is recurrent.

DELIVERY

[0090] RNAi molecules specific for one or more anti-apoptotic and/or multi-drug resistance genes and/or stem cells (e.g., mesenchymal stem cells) transformed to express such RNAi molecules can be directly introduced into, or contacted with, sarcoma (e.g., chondrosarcoma) cells or tissues containing sarcoma cells, or alternatively systemically introduced. RNAi molecules can be introduced into sarcoma (e.g., chondrosarcoma) cells or tissues containing sarcoma cells through a variety of means known to the art, including but not limited to, cationic lipids (see e.g., Yano et al. (2004) Clin Cancer Res 10, 7721- 7726), viral vectors (see e.g., Fountaine et al. (2005) Curr Gene Ther 5, 399- 410; Devroe et al. (2004) Expert Opin Biol Ther 4, 319-327), high-pressure injection (see e.g., Lewis and Wolff (2005) Methods Enzymol 392, 336-350), antibody-assisted endocytosis (see e.g., Song et al. (2005) Nat Biotechnol 23, 709-717), aptamer-chimeras (see e.g., McNamara et al. (2006) Nat Biotechnol 24(8) 1005-1015), polycation targeting ligand (see e.g., Hu-Lieskovan (2005) Cancer Res 65, 8984-8992), and/or modification of the RNAi molecules (e.g., chemical, lipid, steroid, protein) (see e.g., Schiffelers et al. (2004) Nucleic Acids Res 32, e149; Urban-Klein et al. (2005) Gene Ther 12, 461-466; Soutschek et al. (2004) Nature 432, 173-178; Lorenz et al. (2004) Bioorg Med Chem Lett 14, 4975-4977; Minakuchi et al. et al. (2004) Nucleic Acid Res 32, e109; Takeshita et al. (2005) Proc Natl Acad Sci USA 102, 12177-12182).

[0091] Preferably, compositions containing RNAi loaded stem cells are directly introduced directly into tissues containing chondrosarcoma cells, in vivo. For example, a composition comprising transformed stem cells that express RNAi molecules specific for one or more anti-apoptotic genes and/or multi-drug resistance genes, along with pharmaceutically acceptable additives, can be applied topically. Topical application can occur in conjunction with a surgical procedure, so as to apply the composition directly to the chondrosarcoma site. As another example, a composition comprising transformed stem cells that express RNAi molecules specific for one or more anti-apoptotic genes and/or multi-drug resistance genes, along with pharmaceutically acceptable additives, can be injected directly into a chondrosarcoma. As another example, RNAi molecules (e.g., siRNA) can be administered in a composition together with a protamine-Fab (antibody) fusion protein for effective targeted delivery of si RNA in vivo (see Song et al. (2005) Nat Biotechnol 23: 709-17 (2005).

[0092] Anti-apoptotic and/or multi-drug resistance gene silencing agents, such as RNAi molecules, and/or stem cells (e.g., mesenchymal stem cells) transformed to express RNAi molecules can be administered through a variety of routes well known in the arts. For example, compositions comprising RNAi molecules or RNAi loaded stem cells can be introduced via methods involving direct injection (e.g., systemic or stereotactic), implantation of the transfected cells engineered to express RNAi molecules, drug-releasing biomatehals, implantable matrix devices, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, etc.

[0093] Anti-apoptotic and/or multi-drug resistance gene silencing agents, such as RNAi molecules, and/or stem cells (e.g., mesenchymal stem cells) transformed to express RNAi molecules can be administered by controlled- release means or delivery devices that are well known to those of ordinary skill in the art. These include, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, 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 and are within the scope of the invention.

[0094] The RNAi loaded stem cells can be implanted along with a carrier material, such as collagen or fibrin glue or other scaffold materials. Such materials can improve cell retention and integration after implantation. Such materials and methods for employing them are known in the art (see e.g., Saltzman (2004) Tissue Engineering: Engineering Principles for the Design of Replacement Organs and Tissues, Oxford ISBN 01951413OX; Vunjak-Novakovic and Freshney, eds. (2006) Culture of Cells for Tissue Engineering, Wiley-Liss, ISBN 0471629359; Minuth et al. (2005) Tissue Engineering: From Cell Biology to Artificial Organs, John Wiley & Sons, ISBN 352731 1866).

[0095] Compositions comprising RNAi molecules specific for one or more anti-apoptotic genes can be introduced into a subject by a variety of means known to the art. For example, RNAi molecules specific for one or more anti- apoptotic genes can be introduced into a subject via a viral (e.g., lentiviral, adenoviral, retroviral) vector system. Viral vectors provide the additional control of transient or persistent expression of the siRNA. Viral vectors also provide the additional control of using tissue-specific promoters to further improve cell-type specificity. A lentiviral vector system further provides for inducible activation or deactivation of transcription (i.e., conditional RNAi). As another example, RNAi molecules specific for one or more anti-apoptotic and/or multi-drug resistance genes can be introduced into a subject via stable transfection of a cell (e.g., a stem cell) with a plasmid encoding the appropriate sequence from which siRNAs can be transcribed and the subsequent introduction of the transfected cell into the subject.

[0096] Introduction of a composition comprising an RNAi molecule specific for one or more anti-apoptotic and/or multi-drug resistance genes can occur before, during, or after one or more sarcoma treatment modalities, such as surgical resection, radiotherapy, and/or chemotherapy (see generally, Longe, ed. (2005) The Gale Encyclopedia Of Cancer, 2nd edition, Thomson Gale, 1419 p., ISBN-10: 1414403623; Harsh et al., ed. (2003) Chordomas and Chondrosarcomas of the Skull Base and Spine, Thieme Medical Publishers, 372 p., ISBN-10: 0865779856; Doherty (2005) Current Surgical Diagnosis & Treatment, 12th ed., McGraw-Hill Medical, 1490 p., ISBN-10: 007142315X). Introduction of RNAi molecules into a subject can be performed in conjunction with alternate modes of therapeutic treatment, especially where a chondrosarcoma cannot be surgically removed adequately with a wide margin or where chondrosarcoma is recurrent.

FORMULATION

[0097] The composition for delivery of RNAi molecules or RNAi loaded stem cells can further comprise a pharmaceutical carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, albumin, anticoagulants such as CPD (citrate, phosphate, and dextrose), dextran, DMSO, combinations thereof, and the like. The concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the subject's needs. For pharmaceutical compositions and methods of treatment disclosed herein, dosage forms and administration regimes can be determined using standard methods known to skilled artisans, for example as set forth in standard references (see e.g., Remington's Pharmaceutical Sciences, 21 st edition (A.R. Gennaro, Ed.) (2005) Lippincott Williams & Wilkins, ISBN: 0781746736; Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R. C, et al., Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003).

[0098] Compositions described herein can be formulated, for example, for oral, enteral, mucosal, percutaneous, or parenteral administration in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral generally includes subcutaneous, intracutaneous, intravenous, intramuscular, intrasternal, intraarticular, intrathecal, and intraperitoneal. Parenteral infusion or injection is preferred, including continuous infusions or intermittent infusions with pumps available to those skilled in the art.

[0099] Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures and/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 LD₅₀ZED₅₀, where large therapeutic indices are preferred. [0100] If desired, the total desired effective amount 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 dosage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.

[0101] Introduction of compositions comprising RNAi molecules or RNAi-loaded stem cells can occur as a single event or over a time course of treatment. For example, compositions can be administered daily, weekly, biweekly, 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.

[0102] It is to be understood that each citation listed and/or described herein is incorporated by reference.

[0103] Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

[0104] The following non-limiting examples are provided to further illustrate the present invention. 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 invention, 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 invention.

EXAMPLE 1

[0105] The following example illustrates knock-down of anti-apoptotic genes of chondrosarcoma cells using small interfering RNAs (siRNAs). Two well- established Grade Il human chondrosarcoma cell lines were pretreated in vitro with siRNAs that specifically target mRNAs for Bcl-2 (SEQ ID NO: 9; Ambion catalogue no. 42815), Bcl-xL (SEQ ID NO: 9; Ambion catalogue no. 6876) or XIAP (SEQ ID NO: 9; Ambion catalogue no. 2553). The cells were then treated with radiation. The effect of anti-apoptotic gene silencing on cell death was assessed by flow cytometry, while cell survival and proliferation were measured by clonogenic survival assay.

[0106] Results showed that chondrosarcoma cells exhibited radioresistance and increased the expression of Bcl-2, Bcl-xL and XIAP after radiation. When one of Bcl-2, Bcl-xL, or XIAP genes was silenced with the corresponding siRNA, radio-sensitivity increased up to 9.2 fold (p<0.05). When two out of the three anti-apoptotic mRNAs were knocked down at the same time, there was an 1 1.3 fold increase in cell death after radiation (p<0.05).

[0107] Chondrosarcoma Cell Culture

[0108] Two well-establised Grade Il chondrosarcoma cell lines were used. One cell line was JJ012 and the other, SW1353 (American Type and Culture Collection, Manassas, MD). The cells were grown at 37⁰C in a humidified atmosphere that contained 5% CO₂ and 95% air. The culture medium consisted of 40% Dulbecco's modification of Eagle's medium, 40% MEM-α, F-12 (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (Gembio, Woodland, CA), 100 ng/ml human insulin (Lilly, Indianapolis, IN), 25μg/ml ascorbic acid, and 100 nM hydrocortisone (Sigma, St. Louis, MO) (see Schorle et al. (2005) Cancer

Lett. 226, 143-154). The culture medium was changed every three to four days. [0109] RNA Interference Targeting Anti-Apoptotic Gene

[Olio] Apoptotic pathways are regulated by apoptotic and anti- apoptotic gene products (see e.g., Fig. 1 ). Radiation activates the apoptotic pathway. Bcl-2 and Bcl-xL counteract Bax which releases cytochrome c. Cytochrome c activates caspases which are the effectors of apoptosis. XIAP inhibits caspase activation after the release of cytochrome c from the mitochondria. siRNAs were obtained that target Bcl-2, Bcl-xL, and XIAP (Ambion, Austin, TX). Chondrosarcoma cells that were not treated with siRNAs or those treated with non-silencing siRNAs were used as negative controls. Delivery of siRNAs was verified by using FITC negative control siRNAs (Qiagen, Valencia, CA) and X-tremeGENE siRNA transfection agent (Roche, Branchburg, NJ) as a carrier. The intracellular localization of FITC siRNAs was confirmed using florescence microscopy. The siRNA delivery efficiency was 85.7±1.3 % by flow cytometry. The signal strength of each immunoblot band was normalized to GAPDH and quantified using the NIH imaging software.

[0111] Flow Cytometry After Propidium Iodide Staining

[0112] Flow cytometry was performed to verify the effect of radiation. Radiation was carried out at room temperature using a Cs-137 Gamma Cell Irradiator (JL Shepherd & Associates, San Fernando, CA) at an absorbed dose rate of 108 cGy/min. Culture medium was identical in both control and experimental groups. Chondrosarcoma cells were treated with 0, 5, and 10 Gy of γ-rays when the cell confluency had reached approximately 80% in six-well plates. Human embryonal kidney cells (HEK cells) were treated with 0, 5, and 10 Gy of radiation. HEK cells were used as a control to verify the cytotoxic effect of radiation. The dose of radiation was chosen based on previously published report (see Wacheck et al. (2003) Br J Cancer 89: 1352-1357).

[0113] Another set of chondrosarcoma cells were pretreated with small interfering double-stranded RNAs that targeted Bcl-2, Bcl-xL and XIAP 48 hours prior to in vitro radiation. The cells undergoing apoptosis were identified by flow cytometric analysis. Briefly, the supernatant was collected and the cells were washed with Hank's balanced salt solution (HBSS, Invitrogen, Carlsbad, CA); the washing buffer was also collected. After trypsinization of the cells, the plate was incubated for two to three minutes at 37°C. Once all the cells were detached, 2 ml of HBSS containing 5% FBS was added to wash the trypsinized cells. The cells from the six-well plates were collected and placed into their proper tubes and spun down at 1200 rpm for 5 minutes. The cells were fixed with 70% ethanol in a -2O⁰C refrigerator. The cells were resuspended in RNAase buffer, and 10μl of 20 μg/ml propidium iodide (A.G. Scientific, San Diego, CA) was added to the cell suspension. Labeled cells were then counted with a flow cytometer (FACS Calibur; Becton Dickinson Science, San Jose, CA). Monoparametric cytograms of propium iodide fluorescence (FL1 ) versus number of events were created using the Cell Quest (Becton Dickson, NJ) program gating for living cells and excluding dead cells on the basis of their propidium iodide uptake. The results from two different cell lines were analyzed. Each experiment was repeated three times.

[0114] Clonogenic Survival Assay

[0115] Clonogenic survival assay was used to determine the capacity for cell survival and proliferation after radiation (see Banasiak et al. (1999) Radiat. Oncol. Investig. 7, 77-85). After the treatment with siRNA and radiation, 1000 cells from each group were seeded at 60 mm cell culture plate containing the culture medium. Fifteen days later, the cells were stained with crystal violet (Sigma, St. Louis, MO). Colonies larger than 50 cells were counted at low magnification.

[0116] lmmunoblotting

[0117] lmmunoblotting assays were conducted to determine the expression of anti-apoptotic proteins by chondrosarcoma cells and the effect of gene silencing. Briefly, the cells were lysed using buffer IP (10 mM Tris-HCI, pH 7.4, 150 mM NaCI, 1 % Triton X-100, 0.25% Nonidet P-40 and 2 mM EDTA), supplemented with a protease inhibitor cocktail tablet (Roche, Branchburg, NJ).

Equivalent protein extracts (10 μg) from each sample were electrophoresed in A- 20% Tris-Glycine gels (Invitrogen, Carlsbad, CA). The total amount of protein was quantitated with the BCA assay. The protein was transferred to the Immun- Blot PVDF membrane (Bio-Rad, Hercules, CA). The membrane was incubated with Bcl-2, Bcl-xL, XIAP (Cell Signaling, Beverly, MA) and GAPDH antibody (Chemicon, Temecula, CA).

[0118] Statistical Analysis

[0119] Each experiment was repeated three times. The data were expressed as the mean ± standard error (SE) of the mean. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software (version 13; SPSS, Chicago, IL). Differences between each control and experimental groups were analyzed by using one-way analysis of variance between groups (ANOVA/Scheffe), and p < 0.05 was considered statistically significant.

[0120] Results showed that: chondrosarcoma cells are resistant to radiation; anti-apoptotic gene silencing enhances radio-sensitivity; and anti- apoptotic gene silencing with radiotherapy decreases cell survival and proliferation.

[0121] Chondrosarcoma cells are resistant to radiation

[0122] It was tested whether chondrosarcoma cells are resistant to radiation in vitro. Results showed there was no apparent difference of apoptosis rate before and after radiation treatment in chondrosarcoma while control HEK cells underwent extensive cell death. As such, flow cytometric data showed that chondrosarcoma cells are resistant to radiation (see e.g., Figure 2A). Two chondrosarcoma cells showed radio-resistance in comparison to human embryonic kidney cells (HEK).

[0123] Dual gene silencing decreases expression of two different proteins in chondrosarcoma

[0124] In addition, it was determined whether two siRNAs targeting different mRNAs can induce silencing of two different target genes simultaneously. To determine the optimal gene silencing, both time-course and dose-response experiments were performed. Increased expression of Bcl-2, Bcl-xL and XIAP was verified by immunoblotting and the effectiveness of gene silencing determined by siRNAs (see e.g., Figure 2A; Figure 3A; Figure 3B). A decrease in the expression of Bcl-2, Bcl-xL and XIAP proteins one, two and three days after treating chondrosarcoma cells with siRNAs was confirmed (see e.g., Figure 3A). When 1 μg/ml of siRNA was used, there were no significant changes in apoptosis of chondrosarcoma cells (1.04±0.30%) when compared to the negative control (1.01 ±0.28%). Protein expression with and without the siRNA treatment was measured by quantifying the density of immunoblot bands adjusted to GAPDH using the NIH Imaging Analysis software (see e.g., Figure 3B). Target gene knockdown efficiency with 1 μg/ml of siRNA was calculated as: Bcl-2: 65.0±4.5%, Bcl-xL: 79.3±1 .7%, XIAP: 46.8±4.5%. In the same setting, the effect of gene silencing on Bcl-xL and XIAP protein expression increased from day 1 to day 3, while that on Bcl-2 expression partially decreased by day 3 (see e.g., Figure 3A). Dual gene silencing treatment resulted in the down-regulation of two target proteins simultaneously (see e.g., Figure 3B). Silencing of Bcl-2, Bcl-xL, XIAP, Bcl-2 + Bcl-xL, Bcl-xL + XIAP and Bcl-2 + XIAP resulted in a slightly increased fraction of cells undergoing apoptosis. Only dual gene silencing groups showed an up to 3-fold increase in the apoptosis rate in comparison to a non-silencing group (p<0.05).

[0125] Anti-apoptotic Gene Silencing Enhances Radio-sensitivity

[0126] Data indicated that chondrosarcoma cells are resistant to radiation (see e.g., Figure 2A). In addition, it was verified that chondrosarcoma cells overexpress Bcl-2, Bcl-xL, and XIAP in response to radiation (see e.g., Figure 2B). Bcl-2, Bcl-xL and XIAP expressions were verified in two chondrosarcoma cells and were slightly upregulated after radiation. Based on these results, it was concluded that silencing of Bcl-2, Bcl-xL and XIAP mRNAs can enhance radio-sensitivity of chondrosarcoma cells by knocking down the expression of anti-apoptotic gene products that promote cell survival. Further, double-gene silencing can further enhance radio-sensitivity by knocking down two different anti-apoptotic proteins simultaneously. While the untreated chondrosarcoma cells exhibited radioresistance, Bcl-2, Bcl-xL, and XIAP gene silencing enhanced the radio-sensitivity up to 9.2 fold at 5 Gy (p<0.05) and 7.7 fold at 10 Gy (p<0.05) in comparison to the control group on flow cytometric examinations (see e.g., Figure 4A; Figure 4B). In addition, double gene silencing enhanced the radio-sensitivity up to 1 1.3 fold at 5 Gy (p<0.05) and 1 1.2 fold at 10 Gy (p<0.05). Among single gene silencing groups, Bcl-xL was the most effective in enhancing radio-sensitivity. The synergistic effect of double gene silencing on radio-sensitivity was maximal when Bcl-2 and Bcl-xL were knocked down simultaneously.

[0127] Anti-apoptotic Gene Silencing with Radiotherapy Decreases Cell Survival and Proliferation

[0128] Tumor recurrence is an important prognostic factor that negatively affects the clinical outcome following radiation or chemotherapy. Clonogenic cell survival assays were conducted which measure the capacity of cell survival and proliferation.

[0129] Results showed that chondrosarcoma cells can proliferate and form multiple colonies after radiation. When anti-apoptotic genes were silenced in cells that underwent radiation, the number of colonies decreased significantly in comparison to cells without gene silencing (p<0.05) (see e.g., Figure 5A; Figure 5B). Anti-apoptotic gene silencing decreased colony formation up to fourfold at 5 Gy treatment (p<0.05) in comparison to the control group. Combinatorial siRNA treated (Bcl-xL+XIAP) chondrosarcoma cells formed fewer colonies than single siRNA treated and control groups after radiation.

EXAMPLE 2

[0130] The following example demonstrates alteration of gap junctional permeability via co-transfection of Connexin43 (Cx43) (see Lecanda et al. (1998)

MoI Biol Cell 9(8), 2249-2258, incorporated herein by reference). [0131] Cell lines included the osteogenic sarcoma cell line ROS 17/2.8; UMR 106-01 cells; and mouse calvaha osteoblasts MC3T3-E1 . ROS 17/2.8 cells have been shown to express several osteoblastic features, including production of osteocalcin and other matrix proteins. The UMR 106-01 cells were also characterized as having an osteoblastic phenotype. The MC3T3-E1 cell line represents phenotypically immature osteoblasts, derived from spontaneous immortalization of calvaha cells. Culturing of cells was as described in Lecanda et al. (1998).

[0132] Clones of ROS 17/2.8 or UMR 106-01 cells stably expressing either chick Cx45 (ROS/Cx45) or rat Cx43 (UMR/Cx43), respectively, were generated by transfection with the pSFFV-Neo vector containing the reading frames of either connexin. For transient transfections, both these Cx43 and Cx45 constructs in pSFFV-Neo, as well as a construct generated by inserting chick Cx45 in pZeocin (Promega, Madison, Wl), were used.

[0133] The alkaline phosphotase assay; cell proliferation experiments; RNA blots; dye coupling; promoter-luciferase reporter constructs; transient transfection and luciferase assay; immunoblots; and immunofluorescence were each performed/constructed as described in Lecanda et al. (1998).

[0134] The rat osteoblastic cell line UMR 106-01 is poorly dye coupled and expresses predominantly Cx45 and little Cx43 on the cell surface. A clone stably transfected with rat Cx43 (UMR/Cx43) was analyzed by RNA blotting and compared with the parent cells. Basal levels of OC mRNA were very low in this cell line, but they were increased -30% in the UMR/Cx43 transfectants. The OC mRNA levels correlated with higher degrees of coupling and Cx43 abundance in UMR/Cx43, whereas steady-state levels of BSP, OP, and ON were not significantly different in the transfectants, as compared with the parent clones. In contrasting studies for Cx45, transient overexpression of chick Cx45 in MC3T3- E1 cells decreased OC and BSP promoter, while not affecting ON-luciferase activity and upregulating OP promoter activity. [0135] It was next determined whether overexpression of Cx43 in UMR 106-01 cells upregulated the OC promoter, as would be predicted if OC gene transcription were sensitive to the relative expression of Cx43. Similar experiments were also performed in MC3T3-E1 cells, which express endogenous Cx43 but at a lower degree than do ROS 17/2.8, and in the latter cell line. Results showed that basal OC and BSP promoter activities were lower in UMR 106-01 than in ROS 17/2.8 or MC3T3-E1 cells. Co-transfection of OCLUC or BSPLUC with Cx43 increased transcriptional activity compared with vector-transfected cells in both UMR 106-01 (142±16%, and 137±12%, respectively, n = 4) and MC3T3-E1 cells (135±1 1 %, and 1 17±1 1 %, respectively, n = 6), whereas the SV40LUC construct was unaffected. On the other hand, no significant changes in transcriptional activity were observed by transfecting Cx43 into ROS 17/2.8 cells. Thus, the relative expression of Cx43 and Cx45 modulates in a reciprocal manner the transcriptional activity of stage-specific promoters in osteoblasts.

[0136] It was also determined whether transient overexpression of connexin was associated with the predicted changes in dye coupling. It was first demonstrated by immunofluorescence that transfected Cx43 was present preferentially on the cell surface of UMR 106-01 . The proportion of cells exhibiting positive staining was commensurate with a transfection efficiency of -15%, and the higher abundance of Cx43 stain in UMR 106-01 cells reflected expression of both exogenous and endogenous Cx43 in this cell line. Such cellular localization, compatible with functional gap junctions, is identical to that observed in stably transfected clones of the same cell lines. To assess the ability to diffuse dyes, the parachute assay followed by FACS analysis was performed. This method measures the degree of coupling in an entire cell population, expressed as "transfer ratio" (see Ziambaras et al. (1998) J Bone Miner Res 13, 218-228). Thus, if connexin transfection changes gap junctional communication only in a fraction of cells, the contribution of this fraction to average coupling in that population will result in a change of transfer ratio relative to control cells. The transfer ratio of calcein, a negatively charged dye, from donorto acceptor cells was decreased in ROS 17/2.8 cells after transfection with Cx45 (28.8 vs. 33.7 in vector-control cells). The transfer ratio of calcein, a negatively charged dye, from donor to acceptor cells was increased in UMR 106-01 cells transfected with Cx43 (3.4 vs. 2.6 in vector-control cells). These differences are commensurate with a transfection efficiency of -15%. Similar results were reproduced in three different experiments and are consistent with the hypothesis that expression of Cx43 regulates transcriptional activity of osteoblast-specific promoters via changes in gap junctional communication.

[0137] Thus, Cx43, which forms gap junction channels with increased molecular permeability, modulates the expression of specific osteoblastic gene products by regulating the transcriptional activity of their promoters. Overexpression of Cx43 in poorly coupled cells, such as UMR 106-01 , is known to increase gap junctional permeability (see e.g., Steinberg et al. (1994) EMBO J. 13, 744-750). And these changes in gap junctional communication translate into reciprocal modulatory actions on specific osteoblast promoters (see Lecanda et al. (1998)).

EXAMPLE 3

[0138] The following example illustrates chondrosarcoma cells are resistant to chemotherapy. Two well-established Grade Il human chondrosarcoma cell lines were treated in vitro with doxorubicin and the effect on cell death, cell surface expression of P-glycoprotein and doxorubicin uptake were assessed by flow cytometry. Expression of Bcl-2, Bcl-xL, XIAP and P- glycoprotein were measured by immunoblotting.

[0139] Chondrosarcoma and Normal Chondrocyte Cell Cultures

[0140] Two chondrosarcoma cell lines were utilized, chondrosarcoma cell line SW1353 (American Type and Culture Collection, Manassas, MD) and chondrosarcoma cell line JJ012. Two normal articular chondrocyte tissue samples were harvested from patients after obtaining IRB approval. Cartilage specimens were minced with scissors in DMEM (Invitrogen, Carlsbad, CA), producing a cell suspension of small tissue fragments. The suspension was pelleted by centhfugation and the tissue fragments were digested enzymatically in phosphate buffered saline (PBS) containing 1 mg/ml collagenase, 0.15 mg/ml DNAse, and 0.15 mg/ml hyaluronidase (Sigma, St. Louis, MO) for 1 hour at 37° C. The cells were grown at 37⁰C in a humidified atmosphere containing 5% CO₂ and 95% air. The culture medium consisted of 40% Dulbecco's modification of Eagle's medium, 40% MEM-α, F-12 (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (Gembio, Woodland, CA), 100 ng/ml human insulin (Lilly, Indianapolis, IN), 25 μg/ml ascorbic acid, and 100 nM hydrocortisone (Sigma, St. Louis, MO). The culture medium was changed every three to four days.

[0141] Flow Cytometry After P-glycoprotein or Annexin V Staining

[0142] Flow cytometry was used in order to measure cell surface P- glycoprotein expression and drug uptake. The cells were stained with Fluorescence tagged P-glycoprotein antibody (BD pharmingen, San Diego, CA) after harvesting cells with gentle scrapping. The culture conditions were identical for both control and experimental groups. Chondrosarcoma cells and normal chondrocytes were grown in six-well plates and treated with 0, 0.1 , and 1 μM of doxorubicin for 24 hours once a confluency of 80% had been reached. Human embryonic kidney cells (HEK cells) were used as a control to verify the cytotoxic effects of doxorubicin where the dose was chosen based on peak plasma level (1-2 μg/ml or 1 .7-3.4 μM) in patients receiving a standard doxorubicin treatment; see Speth et al. (1987). Flow cytometric analysis was used to identify cells undergoing apoptosis. After trypsinizing the cells, the cells were stained with Annexin V (R&D systems, Minneapolis, MN). Stained cells were then counted with a flow cytometer (FACS Calibur; Becton Dickinson Science, San Jose, CA). Experiments were performed in triplicate.

[0143] Doxorubicin Uptake

[0144] Chondrosarcoma cells were treated with 0, 0.1 and 1 μM of doxorubicin for 24 hours and measured doxorubicin uptake by flow cytometry, since doxorubicin is auto-fluorescent. To measure P-glycoprotein function, the cells were treated with 0.5 μM of doxorubicin for 1 hour and incubated with doxorubicin free media for 1 , 6 and 24 hours. After harvesting cells, doxorubicin level was measured by flow cytometry. Untreated cells were used as a control.

[0145] lmmunoblotting

[0146] lmmunoblotting assays were conducted in order to determine the expression of P-glycoprotein and anti-apoptotic proteins by chondrosarcoma cells and the effect of gene silencing. The cells were lysed using buffer IP (10 mM Ths-HCI, pH 7.4, 150 mM NaCI, 1 % Triton X-100, 0.25% Nonidet P-40, and 2 mM EDTA) supplemented with protease inhibitor cocktail (Roche, Branchburg, NJ). Equivalent protein extracts (10 μg) from each sample were electrophoresed in 4-20% Ths-Glycine gels (Invitrogen, Carlsbad, CA). The total amount of protein was quantified using the BCA assay. The protein was transferred to an Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA) which was then incubated with Bcl-2, Bcl-xL, XIAP (Cell Signaling, Beverly, MA), α-actin (Sigma, St. Louis, MO), P-glycoprotein (Calbiochem, San Diego, CA) and GAPDH antibodies (Chemicon, Temecula, CA). The signal strength of each immunoblot band was normalized to GAPDH and quantified using the NIH imaging software.

[0147] Statistical Analysis

[0148] Experiments were performed in triplicate. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software (version 13, Chicago, IL). Differences between each control and experimental groups were analyzed by using one-way analysis of variance between groups (ANOVA/Scheffe), and p < 0.05 was considered statistically significant.

[0149] Results showed that chondrosarcoma cells exhibited chemo- resistance. Chondrosarcoma cells and normal chondrocytes expressed all proteins examined and treatment with doxorubicin did not significantly change the expression levels. P-glycoprotein expression was found on the cell surface of both chondrosarcoma cell lines. Intracellular doxorubicin concentrations increased in a dose-dependent manner and likewise decreased, after washout, in a time-dependent manner.

EXAMPLE 4

[0150] The following example illustrates knock-down of anti-apoptotic genes and P-glycoprotein in chondrosarcoma cells using small interfering RNAs (siRNAs). Two well-established Grade Il human chondrosarcoma cell lines were pretreated in vitro with siRNAs that specifically target mRNAs for Bcl-2, Bcl-xL, XIAP or P-glycoprotein. The cells were then treated with doxorubicin. The effect of gene silencing on cell death was assessed by flow cytometry, while cell survival and proliferation were measured by clonogenic survival assay.

[0151] Results showed that P-glycoprotein and anti-apoptotic gene silencing enhances doxorubicin sensitivity and decreases cell survival and proliferation following chemotherapy.

[0152] RNA Interference Targeting Anti-Apoptotic Genes

[0153] The siRNAs targeting Bcl-2 (see e.g. SEQ ID NO: 9; Ambion catalogue no. 42815), Bcl-xL (see e.g. SEQ ID NO: 10; Ambion catalogue no. 6876), XIAP (see e.g. SEQ ID NO: 1 1 ; Ambion catalogue no. 2553), and P- glycoprotein (see e.g. Ambion catalogue nos. s10419, s10418, s10420, 1 18137, 4123, and/or 3933) are obtainable from Ambion (Austin, TX). Chondrosarcoma cells that were not treated with siRNAs or those treated with non-silencing siRNAs were used as negative controls. The delivery of siRNAs was verified by using FITC tagged negative control siRNAs (Qiagen, Valencia, CA) and X- tremeGENE siRNA transfection reagent (Roche, Branchburg, NJ) as a carrier, manufacturer's protocol was followed. Transfection was carried out using 1 μg/ml of siRNA for single siRNA treatment groups and 0.5 μg/ml of each siRNA for dual treatment groups to make the total amount of siRNA administered 1 μg/ml. [0154] Chondrosarcoma cells showed fluorescence in the cytoplasm 24 hours after transfection with fluorescence tagged siRNA, verifying the successful introduction of siRNA (see e.g. Figure 10A). To confirm each siRNA effect on target protein, cells were harvested for immunoblotting after siRNA treatment, as described in Example 2 (unless specified otherwise). Target protein expression of each siRNA was significantly decreased in comparison to control groups (see e.g. Figure 10B). Protein expression with and without siRNA treatment was measured by quantifying the density of immunoblot bands adjusted to GAPDH using NIH Imaging Analysis software. The following target gene knock down efficiencies with 1 μg/ml of siRNA were calculated: (SW1353; Bcl-2: 39±5.1 %, Bcl-xL: 72±10.3%, XIAP: 66±12.2%, P-glycoprotein: 47±6.8%) and (JJ012; Bcl-2: 57±10.2%, Bcl-xL: 77±1 1.7%, XIAP: 70±9.6%, P-glycoprotein: 62±9.9%). Chondrosarcoma cells were treated with 1 μg/ml of siRNA and two different concentration of doxorubicin (0.1 μM and 1 μM). Apoptosis rates were then measured to investigate whether or not anti-apoptotic gene silencing would enhance doxorubicin sensitivity in chondrosarcoma cells. While the untreated chondrosarcoma cells exhibited chemo-resistance, Bcl-2, Bcl-xL and XIAP gene silencing enhanced chemo-sensitivity by up to 4.5 fold (p<0.05) at 0.1 μM and 6 fold (p<0.05) at 1 μM in comparison to each control group on flow cytometric examination (see e.g. Figure 1 1 A and B). P-glycoprotein gene silencing also enhanced chemo-sensitivity significantly at 0.1 μM (p<0.05) and 1 μM (p<0.05) in comparison to each control group. Additionally, dual gene silencing enhanced chemo-sensitivity by up to 7.2 fold (p<0.05) at 0.1 μM, 9.9 fold (p<0.05) at 1 μM. Among the single anti-apoptotic gene silencing groups, the knockdown of XIAP was most effective while for the dual gene silencing groups, the synergistic knockdown of Bcl-xL and XIAP was most effective.

[0155] Clonogenic Survival Assay

[0156] The clonogenic survival assay was used to determine the capacity for cell survival and proliferation after chemotherapy ( see Franken et al. (2006), incorporated herein by reference). After the treatment with siRNA and doxorubicin, 1000 cells from each group were seeded onto 60 mm cell culture plates containing the culture medium. Fifteen days later, the cells were stained with crystal violet (Sigma, St. Louis, MO). Colonies larger than 50 cells were counted at low magnification.

[0157] Tumor recurrence is one of the prognostic factors which negatively affects the clinical outcome following radiation or chemotherapy. Clonogenic cell survival assays were conducted and showed that chondrosarcoma cells retain their ability to proliferate and form multiple colonies after doxorubicin treatment (see e.g. Figure 12). When anti-apoptotic genes were silenced in cells which underwent chemotherapy, the number of colonies decreased significantly (p<0.05). The silencing of those genes resulted in a decreased colony formation by up to three fold (p<0.05) at 0.1 μM in comparison to control group. P-glycoprotein gene silencing also showed the significant decrease in colony formation at 0.1 μM (p<0.05). At the highest dose of doxorubicin (1 μM), there was no significant colony formation at all in any of the siRNA treated groups.

EXAMPLE 5

[0158] The following example demonstrates in vitro investigation of human mesenchymal stem cells as a delivery vehicle of siRNA to osteosarcoma cells. Towards the development of targeted siRNA delivery via hMSC (see e.g. Fig 10), it was examined whether transfected hMSCs can pass their siRNA directly to osteosarcoma cells in co-culture.

[0159] Human mesenchymal stem cells derived from bone marrow stroma were acquired from Cambrex (Walkersville, MD). They were cultured according to manufacturer instructions. One day before transfection a 12 well plate was seeded with 40,000 hMSCs/well in 1 ml. of complete MSCBM media with 10% FBS per well (Lonza, Walkersvilee MD). The cells were 50-80% confluent at the time of transfection. hMSCs were transfected with red fluorescent non-targeted siRNA (BLOCK-iT AlexaFuor Red Fluorescent siRNA, Invitrogen, Carlsbad, CA) according to the Lonza hMSC PrimeFect transfection system.

[0160] Twenty-four hours after transfection of red siRNA into hMSC, we co-cultured the hMSCs with GFP tagged osteosarcoma cells that had been growing in complete DMEM media from the ATCC (Manassas, VA). To co- culture the cells, hMSC and GFP tagged OSA were trypsinized, spun down, and resuspended in MSCBM.

[0161] To maximize the probability that the hMSC cells would transfer siRNA to the osteosarcoma cells we co-cultured hMSC and OSA cells in ratios of 1 :1 , 3:1 , 7:1 (see e.g. Fig 1 1 and 12). The plates were monitored for 3 days for cells which fluoresced green and red, indicating GFP-tagged osteosarcoma cells which had been transfected by red siRNA containing hMSC cells.

[0162] Transfection of hMSCs with red-tagged siRNA was successful 86% of the time. This shows that delivery of siRNA into osteosarcoma cells via human mesenchymal stem cells is possible in vitro. It was shown that siRNA can spread within a large synctium of osteosarcoma cells from one cell body to another (see e.g. Fig. 1 1 ).

[0163] As demonstrated above, hMSCs can be used in vivo for targeted delivery of siRNA to osteosarcoma cells, and as valuable cellular vehicles for the delivery of biologic agents for targeting anti-apoptotic genes (see e.g. Fig. 16). This therapeutic approach can avoid the toxicity of systemically distributed anti-cancer agents and contribute to targeted delivery of chemotherapeutic agents to microscopic metastatic centers and increase the survival of osteosarcoma patients.

Claims

CLAIMSWhat is claimed is:

1. A method of increasing sensitivity of a sarcoma to radiotherapy or chemotherapy comprising introducing into a subject in need thereof an effective amount of a composition comprising a transformed stem cell comprising a nucleic acid that comprises an RNA interference (RNAi) molecule specific for a messenger RNA (mRNA) corresponding to at least one gene selected from the group consisting of an anti-apoptotic gene and a multi-drug resistance gene, wherein the transformed stem cell expresses the RNAi molecule.

2. A method of treating a sarcoma resistant to chemotherapy or radiotherapy comprising: introducing into a subject in need thereof an effective amount of a composition comprising a transformed stem cell comprising a nucleic acid that comprises an RNA interference (RNAi) molecule specific for a messenger RNA (mRNA) corresponding to at least one gene selected from the group consisting of an anti-apoptotic gene and a multi-drug resistance gene, wherein the transformed stem cell expresses the RNAi molecule; and exposing the subject to chemotherapy or radiotherapy.

3. A composition for treatment of sarcoma comprising a transformed stem cell comprising a nucleic acid that comprises an RNA interference (RNAi) molecule specific for a messenger RNA (mRNA) corresponding to at least one gene selected from the group consisting of an anti-apoptotic gene and a multidrug resistance gene and a pharmaceutically acceptable carrier, wherein the transformed stem cell expresses the RNAi molecule.

4. The method or composition of any one of claims 1 -3 wherein the sarcoma is a chondrosarcoma resistant to radiotherapy or chemotherapy.

5. The method or composition of any one of claims 1-4 wherein the nucleic acid comprises RNAi molecules specific for mRNAs corresponding to at least two genes independently selected from the group consisting of an anti- apoptotic gene and a multi-drug resistance gene.

6. The method or composition of any one of claims 1 -5 wherein the at least one anti-apoptotic gene is selected from the group consisting of Bcl-2, BcI- xL, and XIAP.

7. The method or composition of any one of claims 1 -6 wherein the at least one multi-drug resistance gene is a P-glycoprotein gene.

8. The method or composition of any one of claims 1 -7 wherein the nucleic acid comprises RNAi molecules specific for mRNAs corresponding to at least one anti-apoptotic gene and at least one multi-drug resistance gene.

9. The method or composition of any one of claims 1 -8 wherein the RNAi molecule is selected from the group consisting of double-stranded RNA (dsRNA), small interfering RNA (siRNA), hairpin RNA (shRNA), multicistronic siRNA, and microRNA (miRNA).

10. The method or composition of any one of claims 1 -9 wherein the RNAi molecule is a small interfering RNA (siRNA).

1 1. The method or composition of any one of claims 1 -10 wherein the RNAi has a length of about 19 nucleotides to about 25 nucleotides.

12. The method or composition of any one of claims 1-1 1 wherein the stem cell is a mesenchymal stem cell.

13. The method or composition of any one of claims 1 -12 wherein the transformed stem cell further comprises a nucleic acid encoding a connexin protein, wherein the transformed stem cell expresses the connexin protein and the connexin protein facilitates transfer of RNAi molecules from the transformed stem cell to a sarcoma cell.

14. The method or composition of claim 13 wherein the nucleic acid further comprises a promoter that is upregulated by expression of the connexin protein.

15. The method or composition of any one of claims 13-14 wherein the connexin protein is a connexin32 protein, connexin37 protein, connexin40 protein, connexin43 protein, or connexin45 protein.

16. The method or composition of claim 14 wherein the nucleic acid further comprises an osteocalcin (OC) promoter or a bone sialoprotein (BSP) promoter, wherein the OC promoter or the BSP promoter is upregulated by the connexin protein, and wherein the upregulation of the OC promoter or the BSP promoter results in increased expression levels of RNAi molecules.

17. The method of any one of claims 1 -2 and 4-16 wherein the subject is a mammal.

18. The method of any one claims 1 -2 and 4-17 wherein the amount of introduced composition comprises about 1 x10⁸ to about 1 x10² of the transformed stem cells.