Development of universal cancer drugs and vaccines

• US 9,394,538 B2
• Filed: 06/02/2010
• Issued: 07/19/2016
• Est. Priority Date: 05/07/2008
• Status: Active Grant

• Current Assignee: David Ts Wu, Shi-Lung Lin
• Original Assignee: David Ts Wu, Shi-Lung Lin
• Inventors: Wu, David T S, Lin, Shi-Lung
• Primary Examiner(s): Schultz, Doug
• Application Number:
  US12/792,413
• Publication Number:
  US 20100240126A1
• Time in Patent Office: 2,239 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2330/51 Specially adapted vectors

Abstract

This invention generally relates to a design and method for developing novel anti-tumor/cancer drugs, vaccines and therapies, using microRNA (miRNA) and its shRNA homologues/derivatives. More particularly, the present invention relates to the use of a nucleic acid composition capable of expressing mir-302-like gene silencing effectors upon delivery into human cells and then silencing mir-302-targeted cell cycle regulators and oncogenes, resulting in an inhibitory effect on tumor/cancer cell growth and metastasis. Mir-302 is the most predominant miRNA found in human embryonic stem (hES) and induced pluripotent stem (iPS) cells, yet its function is unclear. The present invention establishes that in humans mir-302 concurrently suppressed both cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways and eventually blocked over 70% of the G1-S transition. Simultaneously, mir-302 also silences BMI-1, a cancer stem cell marker, and subsequently promotes the tumor suppressor functions of p16Ink4a and p14/p19Arf in inhibiting CDK4/6-mediated cell proliferation. Therefore, the present invention for the first time reveals the tumor suppressor function of mir-302 in humans. This novel finding advances the design and method for developing new cancer drugs, vaccines and therapies directed against multiple kinds of human tumors and cancers, in particular including, but not limited, malignant skin, prostate, breast and liver cancers as well as various tumors.

Claims

1. A method for using a recombinant nucleic acid composition to induce tumor suppression effects in at least a targeted cancer or tumor cell, comprising the steps of:

(a) providing the recombinant nucleic acid composition capable of being delivered and processed to provide at least a gene silencing effector capable of concurrently suppressing the expression of CDK2, cyclin D and BMI-1 oncogene, wherein said gene silencing effector is a hairpin RNA having a sequence consisting of SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15 and SEQ ID NO;

16 and wherein said gene silencing effector can be further processed to provide mir-302a, mir-302b, mir-302c and mir-302d in the targeted cancer or tumor cell; and

(b) treating a cell substrate containing the targeted cancer or tumor cell with said recombinant nucleic acid composition under a condition that the concentration of said gene silencing effector is at least 1.3 fold higher than the natural expression level of mir-302 found in human embryonic stem cell lines H1 or H9 and is sufficient to suppress said CDK2, cyclin D and BMI-1 oncogene;

wherein the cancer or tumor cell is identified as a cell type derived from human neoplastic Tera-2-derived teratocarcinomas.

2. The method as defined in claim 1, wherein said targeted cancer or tumor cell includes a human cell categorized as one of tumor types found in teratoma.

3. The method as defined in claim 1, wherein said recombinant nucleic acid composition includes an expression-competent vector.

4. The method as defined in claim 1, wherein said recombinant nucleic acid composition includes a type-II RNA polymerase-driven promoter.

5. The method as defined in claim 4, wherein said type-II RNA polymerase-driven promoter is inducible.

6. The method as defined in claim 1, wherein said cell substrate expresses mir-302-targeted genes selected from CDK2, cyclin D or BMI-1.

7. The method as defined in claim 1, wherein said recombinant nucleic acid composition further includes a 5′

-donor splice site, an intronic insert site, a branch point motif, a poly-pyrimidine tract, and a 3′

-acceptor splice site.

8. The method as defined in claim 7, wherein said intronic insert site includes at least one of said gene silencing effector.

9. The method as defined in claim 1, wherein said recombinant nucleic acid composition further includes a plurality of exons.

10. The method as defined in claim 1, wherein said recombinant nucleic acid composition is delivered into said cell substrate by a gene delivery method.

11. The method as defined in claim 1, wherein said tumor suppression effects include at least one of cell cycle attenuation or cancer cell apoptosis.

12. The method as defined in claim 1, wherein said tumor suppression effects result from RNA interference.

13. The method as defined in claim 1, wherein said recombinant nucleic acid composition is made by a genetic engineering method.

14. The method as defined in claim 1, wherein said gene silencing effector contains at least a loop structural sequence of SEQ ID NO:

2.

15. A method for using a recombinant nucleic acid composition to induce tumor suppression effects in at least a targeted cancer or tumor cell, comprising the steps of:

(a) providing the recombinant nucleic acid composition capable of being delivered and processed to provide at least a gene silencing effector capable of concurrently suppressing the expression of CDK2, cyclin D and BMI-1 oncogene, wherein said gene silencing effector contains at least a hairpin RNA having a sequence of SEQ ID NO;

3 and consisting of pre-miRNAs of mir-302a, mir-302b, mir-302c and mir-302d, wherein said hairpin RNA is further processed to provide mir-302a, mir-302b, mir-302c and mir-302d in the targeted cancer or tumor cell; and

(b) treating a cell substrate containing the targeted cancer or tumor cell with said recombinant nucleic acid composition under a condition that the concentration of said gene silencing effector is at least 1.3 fold higher than the concentration of mir-302 in human embryonic stem cells H1 or H9 and is sufficient to suppress said CDK2, cyclin D and BMI-1 oncogene;

wherein the cancer or tumor cell is identified as a cell type derived from human neoplastic Tera-2-derived teratocarcinomas.

16. The method as defined in claim 15, wherein said recombinant nucleic acid composition includes an expression-competent vector.

17. The method as defined in claim 15, wherein said recombinant nucleic acid composition includes a type-II RNA polymerase-driven promoter.

18. The method as defined in claim 17, wherein said type-II RNA polymerase-driven promoter is inducible.

19. The method as defined in claim 15, wherein said recombinant nucleic acid composition further includes a 5′

-donor splice site, an intronic insert site, a branch point motif, a poly-pyrimidine tract, and a 3′

-acceptor splice site.

20. The method as defined in claim 15, wherein said recombinant nucleic acid composition further includes a plurality of exons.

21. The method as defined in claim 15, wherein said recombinant nucleic acid composition is delivered into said cell substrate by a gene delivery method.

22. The method as defined in claim 15, wherein said tumor suppression effects include at least one of cell cycle attenuation or cancer cell apoptosis.

23. The method as defined in claim 15, wherein said tumor suppression effects result from RNA interference.

24. The method as defined in claim 15, wherein said recombinant nucleic acid composition is made by a genetic engineering method.

25. The method as defined in claim 15, wherein said gene silencing effector contains at least a loop structural sequence of SEQ ID NO:

2.

26. The method as defined in claim 15, wherein said gene silencing effector consists of sequences of pre-miRNAs of mir-302a, mir-302b, mir-302c, and mir-302d.

27. A method for using a recombinant nucleic acid composition to induce tumor suppression effects in at least a targeted cancer or tumor cell, comprising the steps of:

(a) providing the recombinant nucleic acid composition capable of being delivered and processed to provide at least a gene silencing effector capable of concurrently suppressing the expression of CDK2, cyclin D and BMI-1 oncogene, wherein said gene silencing effector is a hairpin RNA consisting of sequences of pre-miRNAs of SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15 and SEQ ID NO;

16 and is further processed to provide mir-302a, mir-302b, mir-302c and mir-302d in the targeted cancer or tumor cell; and

(b) treating a cell substrate containing the targeted cancer or tumor cell with said recombinant nucleic acid composition under a condition that the concentration of said gene silencing effector is at least 1.3 fold higher than the concentration of mir-302 in human embryonic stem cells H1 or H9 and is sufficient to suppress said CDK2, cyclin D and BMI-1 oncogene;

wherein the cancer or tumor cell is identified as a cell type derived from human neoplastic Tera-2-derived teratocarcinomas.