Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-02-02
2001-08-14
Hauda, Karen M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S008000, C536S023500, C536S024330
Reexamination Certificate
active
06274788
ABSTRACT:
The present invention relates to a bicistronic DNA construct comprising X-myc transgene. In particular, the present invention relates to a bicistronic X15-myc transgene capable of expressing truncated X protein and a full-length murine c-myc protein. More particularly, the present invention relates to a bicistronic DNA construct being an X15-myc transgene for use in the production of transgenic animal model systems for human hepatocellular carcinoma and transgenic animal model systems so produced. The invention is based partially on the discovery that in susceptible transgenic mice that carry a bicistronic X-myc transgene there is an accelerated formation of liver tumors involving all lobes.
BACKGROUND OF THE INVENTION
Hepatocellular carcinoma (HCC) is one of the ten most common human cancers with over 250,000 new cases worldwide each year. Evidence gathered over decades of epidemiological studies clearly indicate that there is an indisputable association between infection due to hepatitis B virus (HBV) and HCC. The incidence of HCC is directly proportional to that of HBV. At least 50% of individuals chronically infected by HBV develop HCC. At present more than 200 million people worldwide are chronically infected. Every year one to two million die as a result of the infection, approximately 700,000 of such deaths being due to HBV associated HCC (Szmuness, 1978, Prog. Med. Virol. 24:40-69).
Although an HBV vaccine exists, the WHO estimates that 400 million people will be chronically infected by HBV by the year 2000. Since, the incubation period for the development of HBV-associated HCC is as long as 30 years or even more, the danger posed by HBV related HCC will continue to remain a major threat for decades. Therefore, there is an urgent need for better therapies to supplement existing ones such as liver resection, transplantation and ethanol injection. Otherwise, the situation is not likely to improve. However, it has been difficult to examine the pathogenic mechanism in great detail because of the limited host range of HBV and the lack of in vitro culture systems to propagate it. In view of this, most of the studies of HCC were, until recently, limited to the analysis of HBV-infected patients and chimpanzees or HBV-related hepadnavirus infections in woodchucks.
The close relationship between HBV and HCC has made it one of the most attractive and useful animal models for exploring the role of viruses in cancer development. The HBV genome has been elucidated and the viral genes implicated in hepatopathogenesis have been characterized. Insertional mutagenesis leading to the activation/inactivation of growth regulatory genes or oncogenes as well as transactivation by viral gene products have been suggested as possible mechanisms of HBV associated carcinogenesis. The integration of HBV DNA does not show site preferences in the human genome. Nevertheless, it has been reported to integrate in the vicinity of some important cellular genes, e.g., cyclin A (Wang et al., 1990, Nature 343:555-557), retinoic acid receptors (Dejean et at., 1986, Nature 322:70-72) and oncogene hst-1 (Hatada et al., 1988, Oncogene 3:537-540). However, in case of woodchuck hepatitis virus, insertional activation of a myc gene has been observed in more than 70% of the liver tumors (Quignon et at., Oncogene 12:2011-2017).
The sequence coding for the X protein appears to play a very important role in the physiological events leading to cell transformation. A majority of patients who are seronegative for HBsAg, on evaluation by RT-PCR for transcripts of HBsAg, HBcAg and HBx, show positivity only for HBx transcripts, clearly indicating that the master molecules of HBV-mediated transformation is HBx (Paterlini et al., Hepatology 21:313-321). It also suggests that the integrated X gene may be important for maintaining the tumor phenotype. Further, HBx has been shown to transactivate a variety of viral and cellular promoters (Caselmann, 1996, Adv. Virus Res. 47:253-302) and modulate the tumor promoting pathways (Kekule et al., 1993, Nature 361:742-745; Chirillo et al., 1996, J. Virol. 70:641-646; Klein et al., 1997, Mol. Cell. Biol. 17:6427-6436). HBx binds the tumor suppressor p53 protein and disrupts the process of apoptosis (Wang et al., 1995, Cancer Res. 55:6012-6012). This action of HBx is found to interfere with the normal surveillance mechanism for removing abnormal cells. Cells with a survival advantage could be selected that in turn may trigger the multi-step process of hepatocarcinogenesis. HBx expression can transform NIH3T3 cells as well as a rodent hepatocyte cell line, FMH202 (Schaefer and Gerlich, 1995, intervirology 38:153-154). However, the cell-based transformation studies using HBx have run into trouble because these cells are quite often reported to lose their immortalized status (S. Schaefer, Personal communication). Thus, it has been extremely difficult to examine the pathogenetic mechanisms of HBV from cell culture studies and there is an urgent need for developing a proper and effective animal model system for such studies.
With the advent of embryo microinjection technology, it became evident that many questions related to HBx-associated pathogenesis might be directly examined by introduction of the X gene into transgenic mice. First, the HBx transgenic mouse line was generated in the outbred CD1 background in which the X gene was introduced under its natural promoter. High level expression of HBx induced progressive changes in the liver beginning with neoplastic lesions, through benign adenomas, and finally to malignant carcinomas that killed most male animals before 15 months of age (Kim et al., 1991, Nature 351:317-320; Koike et al., 1994, Hepatology 19:810-819). Though, these studies demonstrate the oncogenic potential of HBx, others have not observed the induction of HCC in independently developed X gene transgenic mouse strains. (Lee et al., 1990, J. Virol. 64:5939-5947; Perfumo et al., 1992, J. Virol. 66:6819-6823). This discrepancy might be associated with the promoter strength, duration of HBx expression and genetic backgrounds on which the various transgenic models were produced. This is substantiated by the fact that the mice that develop HCC were produced and maintained on CD-1 background which shows a high spontaneous rate of HCC (Homburger et al., 1975, J. Natl. Cancer. Inst. 55:37-45). This might also suggest that HBx might not be sufficient to induce HCC by itself but rather, it functions as a cofactor in the process of hepatopathogenesis. It is therefore, clear that other genetic and epigenetic events and factors are necessary for HCC to develop. In this respect, a significant acceleration of the tumorigenic process was seen in a genetic cross between the HBx transgenic and the WHV/c-myc transgenic mice (Terradillos et al., 1997, 14:395-404), but still not as fast as the pathogenetic studies demanded.
SUMMARY OF THE INVENTION
The present invention relates to a novel bicistronic DNA construct represented as X-myc transgene useful for raising animal models for HCC. In a preferred embodiment, the DNA construct is X15-myc transgene having the potential to express a truncated X protein (X15, having from 58 to 154 amino acids) that encompasses the minimal transactivation domain of HBx (Kumar et al., 1996, Proc. Natl. Acad. Sci. USA 93:5647-5652). In addition, it can express a major form of the full-length murine c-myc protein. The reasons for choosing myc gene were (a) selective amplification of c-myc gene in the HBV related HCC cases (Peng et al., 1993, J. Formos. Med. Assoc. 92:866-870) and (b) frequent activation of both c-myc gene and N-myc gene after integration of the viral DNA (Moroy et al., 1986, Nature 324:276-279; Fourel et al., 1990, Nature 347:294-298). Preferably, the X15 region is positioned 5′ to the murine c-myc gene and is operatively linked to and under the regulatory control of its natural promoter and enhancer I element. The c-myc gene is operatively linked to and driven by the core promoter and enhancer II elements. The construct of the present inventio
Anand Rajesh
Kumar Vjay
Singh Mahavir
Totey Satish
Hauda Karen M.
International Centre for Genetic Engineering and Biotechnology
Ladas & Parry
Shukla Ram R.
LandOfFree
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