Tools and Technologies I
Development of a Novel Targeting and Signal Transduction System for Stem Cell Programming Abstract Cardiovascular disease (CVD) is the leading cause of death in the US with an estimated 60 million patients whose care results in approximately $186 billion in annual costs. There is an urgent need for new therapies for acute and chronic myocardial ischemia (MI) and chronic heart failure (CHF) that often develops subsequent to MI. Stem cell (SC) therapy holds great promise for the repair of damaged heart tissue. However, many fundamental issues relating to repair remain unsolved, such as need to efficiently target the cells to the damaged tissue, integrate the cells into the heart, promote their viability, and stimulate new blood vessel growth. We propose to use the tools from the rapidly evolving field of synthetic biology to build an integrated system to target ES-derived myocardiocytes to infracted myocardium and once there, transduce intracellular signals to drive the expression of effectors to promote cell integration and new blood vessel growth. At the heart of the system are fusion proteins that use either an extracellular single chain antibody or a receptor-binding ligand for targeting and intracellular domains that upon dimerization of two extracellular domains, reconstitute an artificial transcription factor, lexA-VP16 to drive the expression of effector genes that confer appropriate phenotype. This fusion protein and the effectors needed for integration, viability and blood vessel growth will be introduced into cardiomyocytes derived from adult stem cells or embryonic stem (ES) cells. Preliminary efficiency of targeting and repair of damaged myocardium will be assessed. Our system will help improve the efficacy of current stem techniques which are too dependent upon stem cells and their differentiated progeny exhibiting repair behavior that they do not intrinsically possess. ES cells can recapitulate development and adult stem cells have some innate repair capacity, but they do not possess repair or regenerative capacity not already selected by evolution. Our system represents one way to overcome these limitations and engineer appropriate repair behavior. Proof of principle of this prototypical system will not only lead to new therapies for myocardial infarction, but also to the development of a similar systems to repair other damaged tissue by simple substitution of appropriate targeting scFv’s and intracellular effectors.
Statement of Benefit to California:
The development of a universal signaling and target system for ES and adult stem cell programming will enable repair of damaged tissue in patients. ES cells and adult stem cells are limited by evolution in their ability to repair or regenerate tissue. The system and tools provided will create a general framework to extend the capabilities of these cells to enable practical therapies. Californians will benefit by the development of practical therapies based on this framework.
This proposal focuses on the development of an integrated system to target embryonic stem cell (ESC)-derived cardiomyocytes to infarcted myocardium to promote cell integration and tissue repair. The Principal Investigator (PI) proposes to achieve this by first engineering constructs including: 1) a chimeric receptor construct that includes an extracellular single chain antibody variable chain fragment (scFv) that recognizes molecules on damaged myocardium; 2) a chimeric artificial transcription factor construct and 3) reporter or effector constructs under the control of a promoter responsive to the artificial transcription factor. They will then express these constructs in ESC-derived cardiomyocytes and test the targeting and signaling capability of these cells in an in vitro model of acute myocardial infarction. The artificial transcription factor will drive GFP to demonstrate proof-of-principle but would then be used to drive transcription of effector genes to promote cell integration, viability and blood vessel growth. The reviewers found this proposal confusing and had difficulty evaluating its potential impact. They described the proposal as high-risk and raised doubts about its feasibility. The reviewers also questioned whether the principal investigator has the experience or staff required to complete the proposed research. Reviewers were unsure of the potential impact of this proposal but did not feel that a strong rationale was provided, either by preliminary data or clear examples from the literature. One reviewer felt that the value of the “modular gene therapy based systems approach” is vastly overstated in the proposal. The reviewers agreed that this is a high-risk proposal and raised serious doubts about its feasibility. They also criticized its writing and organization, noting that it contained numerous undefined acronyms and unfamiliar terminology. Reviewers felt that the preliminary data was weak, noting that no quantitative data is presented, making it difficult to interpret the significance of the results. One reviewer commented that the cardiomyocyte cell line proposed is not a good model for cardiomyocytes and that primary neonatal cardiomyocytes are the accepted standard for in vitro studies. Reviewers felt that the specific aims are not described in sufficient detail, especially with regard to cell biological methods. For example, in the first aim the applicants state that “each construct will be tested in culture for expression” but fail to describe what assays would be performed or what type of analysis and statistics would be implemented. In the second aim, the proposal jumps from cell line studies to ESC-derived cardiomyocyte studies without any description or preliminary data demonstrating the ability to obtain and culture such cells. One reviewer commented that the applicants base their proposed survival strategy on a single report from the literature and their rationale for the selection of the different factors is not clearly explained or justified with additional literature references. Finally, reviewers thought that the repeated use of the phrase “time permitting” implied that the timeline of the proposed work is not feasible. The reviewers questioned whether the PI has the necessary expertise to carry out this proposal based on his/her biosketch and the research plan. They noted that the research team appears to have little experience working with hESCs. They praised a senior consultant as an excellent cell biology resource and mentor but noted that the 2% listed effort would not be sufficient. Overall, the reviewers found this proposal to be confusing and questioned its impact and feasibility.