M. Yarmush, Rutgers University New Brunswick
The overall goal of the proposed research is to develop effective strategies to increase the stability and titer of retroviral vectors for gene therapy applications. Recently, considerable effort has been dedicated toward utilizing these, and other viral, vectors in a number of clinical trials. Although several publications have described various degrees of success, the clinical success of retroviral vectors is limited by the rapid rate of viral inactivation and limited viral titers. Thus, a retrovirus with enhanced intrinsic stability and high titer would alleviate some of the practical obstacles limiting the success of retroviral vectors in the clinic. It is likely that these principles could also be extended to other viral vectors.
The proposed research seeks to accomplish the following: (1) To determine the mechanisms underlying the loss of retroviral bioactivity and the relative stability of the immature / mature viral particles; (2) To optimize culture conditions and methods, in order to allow the production of high-titer viral vectors; and (3) To modify the viral reverse transcriptase to achieve higher efficiency viral replication. To achieve these goals, physical, chemical, and molecular manipulation techniques will be used with the objective of slowing the rate of retroviral decay and increasing retroviral titer.
The proposed work will provide excellent training for the graduate fellows involved in the project, as well as to students exposed to it through various courses and training programs conducted by the investigators. In addition, the PI will provide an excellent educational environment for various high school and university-wide undergraduate research programs. The scientific approach was designed not only to establish an excellent scientific program, but also to expose students to state of the art technology and methodology through both classroom instruction and direct laboratory interaction. The research itself will provide valuable new information regarding the mechanisms of retroviral decay and methods to address them. The experimental results are likely to have a broad impact on the scientific and clinical venues which utilize or are developing viral vector delivery protocols. In summary, the proposed work seeks to provide fundamental research and a human base of personnel equipped to solve problems related to the implementation of techniques to improve suitability of viral vectors for gene therapy applications.
Retroviral vectors are powerful tools for the introduction of genes into mammalian cells and for longterm gene expression for gene therapy applications. However, since retroviruses degrade rapidly, we sought to determine which components of the retrovirus are responsible for this loss in stability and therefore bioactivity. To this end, we focused on RNA and viral proteins, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. We analyzed the ability of the RNA to direct the synthesis of the viral proteins and demonstrated time-dependent degradation of RNA which correlated with the loss in viral bioactivity. Using a variety of biochemical tools we analyzed the viral proteins and found that the viral proteins did not show any change in secondary structure or evidence of protein degradation. The mechanism underlying the degradation of viral RNA was investigated by generating specific mutations in the RNA sequence, which could ultimately change its stability. These mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus. During the course of the project post-doctoral fellows as well as graduate students received training on the various technical skills needed to assess viral stability. These project members are now prepared to train undergraduate students and additional scientific personnel both in and out of the Rutgers environment. Overall, all personnel on the project obtained exposure to problems in the development, formulation, and delivery of clinical grade molecular therapeutics, and each team member became proficient at designing and conducting experiments in general. As an additional educational approach, RNA expression was assessed using computational approaches. As part of a team with multi-disciplinary scientific approaches, a broad scientific and educational foundation was built.
