Directed complementation

Abstract

A method of producing a tumorigenic mouse cell, the tumorigenicity of which depends on a recombinant gene of interest is disclosed. The method involves: (a) providing a conditionally tumorigenic mouse cell containing a recombinant oncogene operably linked to an inducible promoter, wherein (i) expression of the recombinant oncogene is necessary and sufficient for the tumorigenicity of the tumorigenic mouse cell, and (ii) the inducible promoter is in the uninduced state; and (b) introducing into the cell a recombinant gene of interest that functionally complements the oncogene. Also disclosed is a method of testing a compound for anti-tumor effects. The method includes producing tumorigenic mouse cells the tumorigenicity depends on expression of a recombinant gene of interest, implanting the cells in mice and obtaining tumors from the implanted cells, administering test compounds to the mice, and determining anti-tumor effects, if any, of the compounds.

Description

RELATED APPLICATIONS [0001] This application claims priority from U.S. application Ser. No. 11 / 099,735, filed Apr. 5, 2005.FIELD OF THE INVENTION [0002] The field of the invention is molecular biology, oncology and drug development. BACKGROUND OF THE INVENTION [0003] Mouse models of cancer in which primary tumors are driven by specifically engineered oncogenes have been increasingly useful tools in cancer research in recent years. Although they require relatively long timeframes for genetic crosses and latency of spontaneous tumors, current methods of producing such genetically engineered mice generally have been adequate for producing relatively small numbers of genotypes for basic research. [0004] Primary tumors from mouse models would be particularly useful in drug development studies, as well as in basic research. This is because similar or indistinguishable tumor phenotypes, e.g., breast carcinoma, can undergo neoplastic transformation through the spontaneous acquisition of dif...

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Benefits of technology

[0022] A notable advantage of the present invention is that when a single source of conditionally tumorigenic cells is used to generate different lines of primary tumor material by introducing different genes of interest, the effects of the different genes of interest on the tumors, with and without drug treatment, can be evaluated in exactly the same genetic background. In contrast, if separate mouse models were independently engineered to incorporate the same genes of interest and spontaneous tumors were generated, the genetic background of the gene of interest would be different in each model. Consequently, the type of comparison possible with the present invention would not be possible using the separately engineered models.

[0023] Looking at the genetic background question in the opposite way, a given gene of interest can be introduced separately into conditionally tumorigenic cells from multiple tumors. This will allow the effect of a given gene of interest, with and without drug treatment, to be evaluated in different genetic backgrounds.

[0024] For any given conditionally tumorigenic cell, the inducible recombinant oncogene is known. With knowledge of the inducible recombinant oncogene in hand, the skilled person can identity one or more genes of interest that will functionally complement the inducible recombinant oncogene. For example, the receptor tyrosine kinase, Her2 / Neu / ErbB2 is known to be important for the viability of a subset of human breast cancers. Much is known about the downstream mediators of Her2, and this information has been summarized by publicly available sources, e.g., Biocarta. From such information, the skilled person can predict a useful number of complementing genes with a reasonable expectation of success. For example, genes expected to complement Her2 would include ErbB1 / EGFR, ErbB3, PLK3, KIRREL, PI3K, Ras, Akt, Raf, Erk1 and Erk2.

[0025] One of the genes expected to complement Her2 is the ErbB3 gene, also known as HER3, MDA-BF-1 and MGC88033, which encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. The ErbB3 membrane-bound protein has a neuregulin binding domain but not an active kinase domain. It therefore can bind the neuregulin ligand but not convey the signal into the cell through protein phosphorylation. However, it does form heterodimers with other EGF receptor family members which do have kinase activity. Heterodimerization leads to the activation of pathways which lead to cell proliferation or differentiation. Amplification of this gene and / or overexpression of its protein have been reported in numerous cancers, including prostate, bladder, and breast tumors (see, e.g., van der Horst et al., 2005, Int J Cancer 115:519-527; Holbro et al., 2003, Proc Natl Acad Sci USA. 100:8933-8938).

[0026] Another gene expected to complement Her2 is the gene designated PLK3 (polo-like kinase 3), also known as CNK, FNK, PRK, and 1—44702099GP201. The PLK3 gene encodes a putative serine / threonine kinase and is a member of the “polo” family of serine / threonine kinases, which is likely to play a role in cell cycle progression and tumorigenesis (see, e.g., Li et al., 1996, J Biol Chem 271:19402-19408). High expression of PLK3 has been detected in cancers of the bladder, breast, colon, ovary, pancreas and lung (see, e.g., Dai et al., 2000, Genes Chromosomes Cancer 27:332-336; Li et al., 1996, J Biol Chem 271:19402-19408).

[0027] Another gene expected to complement Her2 is the gene designated KIRREL, also known as NEPH1, FLJ10845, LOC348416 or 1—154843723, which encodes a protein member of the nephrin-like protein family and contains an immunoglobulin domain. KIRREL expression is elevated in chondrosarcoma, glioblastomas, including glioblatomas expressing mutant activated EGFR, astrocytomas and medulloblastomas, pancreatic adenocarcinoma, breast carcinomas and colon adenocarcinoma, and melanomas.

Problems solved by technology

Consequently, conventional xenograft experiments using an established human cancer cell line are likely to provide an incomplete picture.

In spite of the desirability of genetically defined primary tumor material for drug development studies, the time and resources that would be required to generate the necessary mouse models have made it impractical to design preclinical drug development studies around the use of primary tumor material in which tumorigenicity depends on a pre-selected gene of interest, i.e., target gene.

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