Development Project 4

Project 4
Assessment of the Efficacy of Novel Adenovirus-based Gene Therapy in Urogenital Cancers

Investigators and Areas of Expertise:

Jer-Tsong Hsieh, Ph.D., and Kenneth Koeneman M.D., Department of Urology, UT Southwestern will be responsible for generating recombinant adenoviruses and in vivo animal model carrying human urogenital tumors. Drs. Mason, Constantinescu and Oz, Department of Radiology, UT Southwestern, will be responsible for all labeling and quality control of the labeled product and all the imaging studies proposed in this project.

Hypothesis and Specific Aims:

Today, cancer gene therapy has become a more realistic approach because rapid advances in molecular genetic techniques have revealed the alterations of cellular oncogene and/or tumor suppressor genes that occur in cancer cells. The discovery of common alterations associated with many cancer types may reveal blueprints for developing clinical care in a cancer-specific manner (Research Area 2- Genetic Signatures). In addition, the increasing number of tissue-specific genes discovered could result in subsequent cloning of their promoters that aid in further design of expression vectors with a tissue-specific suicide gene therapy. Several practical and theoretical considerations make recombinant adenovirus an attractive vector for cancer gene therapy. For urogenital tumors, such as prostate and bladder cancers, we and others demonstrated that recombinant adenovirus appears to be an effective gene therapeutic vector to deliver exogenous DNA into some of these cancer cells using in vivo animal models. However, several issues need to be resolved prior to clinical application. For example, patient selection, the optimal dosage for patients, and monitoring tumor response. Therefore, we believe that determining viral distribution, uptake and transgene expression using non-invasive imaging techniques will provide critical experimental pre-clinical data and ultimately be beneficial for patients.

We recently reported that there is a heterogeneous expression of a high affinity adenoviral receptor (CAR) among urogenital tumors. Using both the monoclonal antibody against CAR and reverse transcription-polymerase chain reaction, we also demonstrated that CAR levels in various cell lines correlate with viral sensitivity. Similar results were reported in two other cancer types: glioma and melanoma. Taken together, CAR may represent a surrogate marker for viral sensitivity of any target cell.

We believe that non-invasive imaging techniques will provide a unique opportunity to facilitate the basic research for cancer gene therapy and to develop a potential agent for monitoring the outcome of gene therapy. The outcome of this study will help us to optimize the viral dosage and achieve better therapeutic index.

Hypothesis: CAR (cocksackie adenovirus receptor) is a unique marker for selecting suitable patients for gene therapy.

Specific Aims: 1) Establish in vivo tumor imaging system to monitor human tumor growth in orthotopic and bone animal models.
2) Evaluate CAR as a surrogate marker for the human urogenital cancer gene therapy using an orthotopic animal model.
3) Monitor in vivo transgene expression of adenovirus and tumor response in an orthotopic animal model.
4) Determine the CAR expression levels in the patients with urogenital cancer.

Background and Significance:

With the advent of PSA screening, many more men are identified early for risk of prostate cancer. This has generated the major issue of who requires urgent aggressive therapy and who will benefit from “wait and see”. Most therapies (radical prostatectomy, external beam radiation, hormone ablation, cryotherapy, and brachytherapy) can have severe impact on quality of life with potential impotence and incontinence, even for the best surgeons and Centers. Thus, there is a search for alternative therapies and prognostic markers to differentiate tumors types with greater specificity. For diagnosis, several groups have shown that MR spectroscopy of suspicious lesions can differentiate benign from malignant tissues on the basis of choline, creatine and citrate concentrations. This may also be valuable in assessing the extent of tumors to assist IMRT (intensity modulated radiotherapy) planning while sparing uninvolved tissue (bladder and rectum). This can help direct biopsies and may ultimately provide rigorous prognosis. We intend to survey patients using MR spectroscopy and physiological imaging (Core 1) prior to biopsy to seek powerful correlations with pathologic/histologic findings. Gene therapy is an innovative approach to the treatment of malignant and benign disorders characterized by genetic alterations that may serve as therapeutic targets. In addition, cytotoxic genes such as HSV-TK and CD-HSV1 (cytosine deaminase) and cytokines for boosting host immunity are good candidates for cancer gene therapy. While early gene therapy has focused on direct intra tumoral injection of replication incompetent virus, a systemic approach offers potential benefit particularly in the case of non-localized disease. However, some essential issues related to the efficiency of virus transfection and expression and possible side effect have not been addressed. For example, since viral proteins are known to be cytotoxic and immunogenic, repeated administration of adenovirus may elicit an immune response from the host. It is known that viral-induced inflammatory response is often involved in the cellular mediated immunity, that is, infiltration of CD4/8⁺ T cells. Thus, any agent that can increase the infectivity of recombinant adenovirus at a lower viral dosage may minimize any adverse side effects.

A high affinity receptor (i.e., CAR) for adenovirus type 5, which is frequently used as a viral vector for gene therapy, has been cloned. Sequence analysis indicates that this adenoviral receptor CAR cDNA encodes a typical immunoglobulin (Ig)-like membrane protein with two Ig domains that may interact with adenovirus fiber protein. Our investigations of clinical specimens indicate reduced CAR mRNA expression in tumor biopsy only and not in normal tissue from the same patient. We have optimized the immunostaining protocol using monoclonal antibody against CAR on paraffin-embedded archival tissue. Our data indicated that CAR protein is only expressed in the transitional epithelial cells, suggesting that CAR may be a useful imaging marker not only for viral distribution, but also for tumor lesions themselves. Therefore, clinically, CAR can be a potential screening marker for selecting a patient population suitable for gene therapy, and a surrogate marker for monitoring the distribution of viral administration.

References:

13. Gleave, ME, Hsieh JT, Wu HC, von Eschenbach AC, Chung LW. Serum prostate specific antigen levels in mice bearing human prostate LNCaP tumors are determined by tumor volume and endocrine and growth factors. Cancer Res. 1992, 52, 1598-605.

14. Hall, MC, Li Y, Pong RC, Ely B, A. S, Hsieh JT. The growth inhibitory effect of p21 adenovirus on human bladder cancer cells. J. Urol. 2000, 163, 1033-1038.

15. Kleinerman, DI, Dinney CP, Zhang WW, Lin SH, Van NT, Hsieh JT. Suppression of human bladder cancer growth by increased expression of C-CAM1 gene in an orthotopic model. Cancer Res. 1996, 56, 3431-3435.

16. Li, Y, Pong RC, Hall MC, A. S, Tseng C-P, Wang Z, J. B, Hsieh JT. Loss of adenoviral receptor expression in human bladder cancer cells- A potential impact on the efficacy of gene therapy. Cancer Res. 1999, 59, 325-330.

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Thursday, September 20, 2001

Southwestern In Vivo Cellular and Molecular Imaging Program