MULTICISTRONIC siRNA CONSTRUCTS TO INHIBIT TUMORS

Inactive Publication Date: 2011-09-29
THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]Multicistronic short interfering RNA constructs include two or more self-complementary sequences targeting a plurality of genes encoding for example, a sequence encoding human urokinase-type plasm

Problems solved by technology

As such, prostate cancer poses a major public health problem in the United States and worldwide.
Glioblastoma multiforme (GBM) is a highly malignant primary central nervous system neoplasm, which is highly refractory to therapy.
Therapy which affects normal brain tissue, is not acceptable.

Method used

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  • MULTICISTRONIC siRNA CONSTRUCTS TO INHIBIT TUMORS
  • MULTICISTRONIC siRNA CONSTRUCTS TO INHIBIT TUMORS
  • MULTICISTRONIC siRNA CONSTRUCTS TO INHIBIT TUMORS

Examples

Experimental program
Comparison scheme
Effect test

example 1

Endogenous uPA and uPAR Protein Expression is Associated with In Vitro Invasiveness of Human Prostate Cancer Cells

[0104]PC3 cells are highly metastatic, whereas DU145 and LNCaP cells are moderately and poorly metastatic, respectively. uPA and its receptor uPAR are involved in tumor invasion and metastasis. The levels of these proteins in the three human prostate cancer cell lines with different metastatic potentials were compared. As shown in FIG. 1A, uPA and uPAR protein levels were significantly higher in PC3 and DU145 cells as compared with the poorly metastatic LNCaP cells, which expressed undetectable levels of these proteins. A similar trend was seen in uPA activity as assessed by fibrin zymography (FIG. 1B). Thus, uPA and uPAR protein levels as well as uPA activity were positively correlated with their known metastatic potential. The ability of the prostrate cancer cells to invade Matrigel, a gel layer composed of basement membrane proteins, was examined. This assay is a well...

example 2

Efficient Knockdown of uPA and uPAR Gene Expression in Human PC3 Prostate Cancer Cells Using RNAi

[0105]Biological role of uPA and uPAR in prostate tumor progression was investigated using small hairpin RNAs to knockdown endogenous uPA and uPAR gene expression in the human prostate cancer cell line PC3, which expresses uPA and uPAR as well as a high metastatic potential. pcDNA3-CMV vectors were developed containing small hairpin constructs capable of generating 19 or 21-nt duplex RNAi oligonucleotides corresponding to either uPA or uPAR. Also, a single bicistronic construct driven by cytomegalovirus (CMV) promoter to deliver dual small hairpins targeted against both uPA and uPAR was constructed to test the effectiveness of simultaneously inhibiting expression of two endogenous genes (FIG. 2A). The vectors expressing shRNAs for uPA, uPAR and the uPA-uPAR combination were transfected into PC3 cells.

[0106]As shown in FIG. 2B, analysis of the shRNA-transfected cells for uPA and uPAR expr...

example 3

Knockdown of uPA and uPAR Expression by RNAi Inhibited Matrigel Invasion of PC3 Cells

[0109]One of the functions of uPA and uPAR is promotion of invasion, a process necessary for tumor metastasis. The impact of uPA and uPAR knockdown on PC3 cellular invasion was evaluated by a Matrigel invasion assay using the shRNA-transfected cells. When compared with mock cells or cells transfected with EV / SV, sh-uPAuPAR-transfected cells showed a substantial reduction in invasive capacity (FIG. 3A). Invasion of PC3 cells was reduced to 75% of that of the controls (i.e., mock or EV / SV-transfected cells) by sh-uPA and to 90% by sh-uPAuPAR (FIG. 3B). Although knockdown of uPAR alone did not show a significant decrease in invasion, knockdown of uPA as well as uPAR had a significant effect (FIGS. 3A & 3B), suggesting that PC3 cell invasion into matrigel is substantially regulated by coordinated function of uPA and uPAR. These results show that uPA and uPAR expression is required for prostate cancer in...

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Abstract

Multicistronic short interfering RNA constructs targeting in various combinations a human urokinase-type plasminogen activator receptor (uPAR), human urokinase-type plasminogen activator (uPA), human matrix metalloprotease 9 (MMP-9) and cathepsin B (CB) inhibit tumors.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a Continuation of co-pending patent application Ser. No. 11 / 748,733, filed May 15, 2007, which is a Continuation-in-Part of International Application No. PCT / US2005 / 41709, filed Nov. 17, 2005, which claims priority to U.S. Ser. No. 60 / 629,659, filed Nov. 18, 2004. The disclosures set forth in the referenced applications are incorporated herein by reference in their entireties.STATEMENT OF GOVERNMENT SUPPORT[0002]The government has rights in the disclosed invention due to partial support of NIH / NCI Grants: CA 85216, CA 75557, CA 76350, CA 92393, CA 95058, CA 116708 and NIH / NINDS Grant: NS 47699 and NS 57529.BACKGROUND[0003]Tumor progression involves modulation of tumor-cell adhesion during cell migration and degradation of the extracellular matrix (ECM) during tissue invasion. An intricate balance of proteases, their activators and their inhibitors, regulates both these processes during tumor invasion. Three classes of ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/09C07H21/02C12N15/113
CPCC12N15/1137C12N15/1138C12Y304/21073C12N2310/14C12N2310/111A61P35/00
Inventor RAO, JASTI S.GONDI, CHRISTOPHER S.LAKKA, SAJANI S.
Owner THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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