Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
1999-12-17
2003-07-22
Wehbe′, Anne M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C800S008000, C800S281000, C800S320100, C800S281000, C800S281000, C800S281000, C800S008000
Reexamination Certificate
active
06596924
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a graft animal model for propagating HPV and for evaluating and testing candidate therapeutic agents against HPV. The animal model comprises, a recipient animal engrafted with injured skin graft infected with a host-specific papilloma virus (PV). The grafted skin, having demonstrable papillomas supports the propagation of its host-specific PV. The invention particularly relates to a highly reproducible xenograft animal model for hosting and propagating human papillomavirus, thereby providing a means for generating infectious human PV suspensions and for passaging papillomavirus. The invention additionally relates to a novel method for generating the xenograft human animal model.
BACKGROUND OF THE INVENTION
Papillomaviruses (PV) are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been recognized in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types papillomavirus (HPV) that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincott—Raven Pub., Philadelphia, N.Y.).
There are more than 25 HPV types that are implicated in anogenital diseases, these are grouped into “low risk” and “high risk” types. The low risk types include HPV type 6, type 11 and type 13 and induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States there are 5 million people with genital warts of which 90% is attributed to HPV-6 and HPV-11. About 90% of SIL are also caused by low risk types 6 and 11. The other 10% of SIL are caused by high risk HPVs.
The high risk types papillomaviruses are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and -18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide (Fields, 1996, supra).
Treatments for genital warts include physical removal such as cryotherapy, CO
2
laser, electrosurgery, or surgical excision. Cytotoxic agents may also be used such as trichloroacetic acid (TCA), podophyllin or podofilox. Immunotherapy is also available such as Interferon or Imiquimod. These treatments are not completely effective in eliminating all viral particles and there is either a high cost incurred or uncomfortable side effects related thereto. In fact, there are currently no effective antiviral treatments for HPV infection, since with all current therapies recurrent warts are common (Beutner & Ferenczy, 1997, Amer. J. Med., 102(5A): 28-37).
The life cycle of HPV is closely coupled to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. Unlike normal cells, cellular division continues as the cell undergoes vertical differentiation. As the infected cells undergo progressive differentiation the viral copy number and viral gene expression increase, with the eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles (Fields, 1996, supra).
Papillomaviruses are fastidious viruses that cannot be propagated in vitro. As such, the virus requires a host-specific animal for growth. The ineffectiveness of the current methods to treat PV infections has demonstrated the need to identify new therapeutic agents as a means to prevent and treat HPV infections. The success of developing candidate therapeutic agents to combat papillomavirus has been limited in part due to difficulties including, propagating the virus, obtaining sufficient infectious viral particles and the lack of a good in-vivo model to evaluate the effectiveness of candidate therapeutic agents. Attempts to overcome these difficulties have been addressed by generating xenograft animal models for human papillomavirus. However, all the models known in the prior art have had limited success in overcoming these difficulties.
The ideal animal model is described as having the following attributes: being widely available, easy to handle and maintain in a laboratory, large enough to provide tissue samples, able to induce and form papilloma lesions that are comparable to those in humans, the papillomas should be readily accessible for treatment, and able to yield a large amount of infectious viral particles (Stanley, et al., 1997, Antiviral Chemistry & Chemotherapy, 8(5):381-400).
In U.S. Pat. Nos. 4,814,268 and 5,071,757 (Kreider et al.), human skin tissue subjected to human papillomavirus was grafted under the renal capsule of athymic mice. This is a complex procedure which requires surgical refinement. The graft is allowed to remain in the animal until recoverable quantities of the virus are produced. Examination of the graft site and recovery of viral particles requires the animals to be killed. The infectivity of the recovered viral particles from the graft site was reported to be only at a 10
−2
dilution. More importantly, since the papillomas formed are not visible, evaluation of therapeutic agents necessitates sacrificing the animal. Subsequent attempts by this group (Kowett et al., 1990, Int. Virology, 31:109-115) to replicate these published results, harvesting infectious viral stock capable of infecting other animal models, have failed. The authors hypothesized that the first wart tissue collected from patients and used to infect an animal model probably contains more infectious virions and is thus successful in initiating papilloma infection in the xenograft animal.
Bonnez W. et al. (1993, Virology 197:455-458) described human foreskin infected in vitro with HPV type 11, implanted under the renal capsule, peritoneum and subcutaneous in SCID mice. Only 58% of the grafts showed signs of HPV infections. In the subcutaneous implanted grafts, only 25% were positive for HPV by immunocytochemistry and RT-PCR. The resultant subcutaneous papillomas were not serially passaged or harvested.
Brandsma J. L. et al. (1995, J. of Vir. 69:2716-2721) and U.S. Pat. No. 5,811,632, describe the delivery of HPV type 16 genomic DNA to human foreskin engrafted onto SCID mice. In total 16 grafts were inoculated with naked HPV DNA, eight inoculated pre-engrafting and eight post-engrafting. Only two grafts inoculated post-grafting appeared to develop signs of HPV infection. However these two prior art documents do not teach harvesting infectious viral particles or the passaging of papillomavirus.
Sexton C. J. et al. (1995, J. of Gen. Vir. 76:3107-3112) described a grafting method whereby a glass cover slip was first inserted into the graft site of a SCID mouse for one to two weeks. This is replaced with a silicone grafting chamber in which benign wart tissue was placed. After five weeks, macroscopic warts developed. Attempts to graft the wart tissue resulted in hyperproliferative human epithelium devoid of viral infection. Thus serial passaging of these warts and harvesting infectious particles are not taught.
Bonnez W. et al. (199
Boehringer Ingelheim (Canada) Ltd.
Devlin Mary-Ellen M.
Pocchiari Susan K.
Raymond Robert P.
Wehbe′ Anne M.
LandOfFree
Graft animal model for high induction of papillomas, the... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Graft animal model for high induction of papillomas, the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Graft animal model for high induction of papillomas, the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3092623