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Directed Delivery of RNA Interference Strategies to Breast Malignancies
Title: Directed Delivery of RNA Interference Strategies to Breast Malignancies
In theory, gene therapy offers an attractive approach to treating breast tumors as well as other types of malignancies. Unfortunately, for practical use, this type of therapy requires a degree of targeting and selectivity on the part of the gene delivery agent that has yet to be achieved in a form suitable for widespread clinical use. In their current state of development, none of the available viral delivery systems has proven ideal for such application, with key technical shortcomings posed by each agent. The fundamental shortcomings of most gene therapy schemes have been inadequate targeting of the gene vector to the cell type desired, and insufficient potency of the agent being delivered. Transduction efficiencies with typical delivery vehicles in tissue are insufficient in most instances for transduction of a majority of malignant cells. As a result, transgene encoded proteins are frequently either generated in inadequate quantities to have a major impact, or in the wrong locations. Alternatively, using other systems, efficiency is high but too indiscriminate, resulting in expression of foreign gene sequences in healthy cells, often triggering severe host immune responses. These considerations have complicated the picture considerably with respect to the development of successful anti-tumor gene therapy strategies. Fortuitously, recent developments promise to overcome these limitations.
Recent advances in viral vector technology offer the means to remove the obstacle of insufficent specificity of the viral agent. As a small, nonpathogenic human parvovirus, gene vectors derived from Adeno Associated Virus (AAV) offer unique advantages for clinical applications of gene therapy. Recent insights into the capsid structures of these small non-enveloped viruses make it practical to engineer specific modifications and restrictions to their inherent broad host cell tropisms. By combining this technology with new information regarding receptors and other molecules suitable for targeting on the surfaces of breast tumor cells, it becomes practical to custom design viral vectors which will selectively home to breast malignancies. The parallel emergence of RNA interference (RNAi) technology offers the opportunity to overcome the other shortfall of many gene therapy strategies – insufficient potency of the agent being delivered. Both of these developments are combined in this project to derive a set of gene vehicles with high selectivity as well as potency against breast malignancies.
c. Specific Aims
1. Construction of AAV Capsids With Specificity.for Breast Tumor Cells
2. Development of RNA Interference Strategies Against Breast Malignancies
3. Evaluation of Targeted Delivery of RNA Interference Upon Breast Tumors in Animal Models.
Our immediate aims in this project are to engineer a set of targeted AAV vectors, and test their potency in well-established murine breast cancer models. Our evaluation will be comprised of two parts; first, with regard to the specificity of the engineered vectors for the tumors, and second, with regard to the efficacy as anti-tumor agents of the interfering RNAs encoded in these vectors. While our short term goal is focused on a specific strategy, successful targeting of viral vectors to breast tumors will have wide ranging ramifications for using recombinant AAV to target breast cancer, as well as other types of malignancies, by many different genetic strategies.
e. Potential Outcomes and Benefits of the Research
This study will evaluate the role of a set of targeted gene therapy vectors as a novel and powerful addition to the armory available for breast cancer treatment, bringing unique advantages over more conventional approaches. This proposal is highly innovative both in terms of the gene delivery vehicles deployed, and the genetic agents to be delivered. However, while they represent new approaches, they are built directly upon a solid foundation of previous work in vector design, as well as the biology of breast tumors, and are ideas with a strong conceptual grounding. We have every reason to believe treatment with the gene therapy agents will greatly slow progression of the illness, if not reverse it. Tumor size and number, extent of apoptosis of tumor cells, vessel density, and survival can all be measured accurately in the mouse models. We predict the gene vectors delivering siRNA will result in reproducible increases in apoptosis of tumor cells, and reductions in tumor volume and number of metastases. The efficacy of the novel agents in this study against breast cancer can also be compared directly with animal studies from previous trials in the same mouse models, as a quantifiable index of success. Because key aspects of our strategy have been designed to greatly enhance the efficiency and selectivity of gene delivery, without trying to exceed the practical limits of the technology, we believe its likelihood of success is high. We are confident about the potential of this project to lead to clinical trials of novel therapies targeting breast cancer in a short timeframe.
Title: Directed Delivery of RNA Interference Strategies to Breast Malignancies
Breast cancer is the most common malignancy among women, and the leading cause of death in nonsmoking women. In 2000, approximately 182,800 new cases of breast cancer were diagnosed in this country, with an estimated 40,800 deaths from the disease (Greenlee et al, 2000). Gene therapy holds great promise to treat breast cancer and other malignancies. A great deal is now known about the biology of breast tumors; as a result, specific key points have been identified at which the tumor biology could potentially be disrupted, if new gene sequences could be delivered into the tumor cells. In theory, viruses carrying a toxin gene or other anti-cancer agent could be used to home in on tumor cells, sparing healthy tissue. Because of their small size, viruses carrying anti-cancer genes should be able to reach even small far flung metastases from diffuse, late stage tumors This would offer major advantages over traditional treatments, which expose healthy tissues as well as tumor cells to toxic chemicals or radiation, and rely upon the relatively greater susceptibility of tumor cells for their efficacy. The principal stumbling block to taking this technology out of the lab and into widespread clinical use has been deficiencies in the available virus vectors. The most serious shortcoming has been inadequate targeting, i.e. the virus vectors are not specific for the tumor cells. Most of the viruses evaluated either do not transduce tumor cells well, or also readily infect healthy cells in the surrounding tissue, causing injected gene therapy agent sto be quickly diluted, and undermining the strategy of delivering the anti-tumor agent only to the malignancy. A related key issue has been the potency of the anti-tunmor agent itself, which becomes more critical when the effective dose of the agent reaching the tumor cells is low.
Vectors derived from a harmless human virus, Adeno Associated Virus (AAV), are one of the best available candidates, by virtue of their small size, the fact that AAV does not itself cause any disease, and does not provoke an over-response by the patient’s immune system. AAV vectors also enter tumor cells efficiently, including cells from breast tumors, as well as the cells which line their blood vessels. However, standard AAV vectors also enter many other kinds of cells, including healthy blood vessels.
Recently a number of short peptides have been identified which home to and bind almost exclusively to proteins present on the surfaces of breast tumor cells, and are not widespread elsewhere in healthy tissue. This information can be combined with new knowledge about the structure of the AAV virus to create engineered viruses that will home in on tumor sites. We propose to use tumor-targeted viruses to deliver a new and highly promising new class of potent anti-tumor agent, short interfering RNAs (siRNA) which can switch off particular genes essential for the tumor to grow and sustain itself. Because these viruses work as well in animal cells as in human cells, the strategies can be tested directly in animals, as a valuable prelude to clinical studies. Success with this approach should quickly lead to human trials of these siRNA, as well as enable other promising gene therapy strategies to interfere with the biology of breast tumors to be targeted to these malignancies in engineered virus particles.