One common form of cardiotoxicity associated with drugs is an electrical abnormality known as induced long QT syndrome (LQTS). Due to the risk of progression from LQTS to life-threatening irregular heart rhythms, the pharmaceutical industry devotes much effort to eliminate LQTS-inducing compounds early during the drug development process. One way to try to predict the risk of drug-induced LQTS in humans is by animal models or by using cell-based assays. Animal models may or may not accurately reflect the biology of humans, and while use of animals in drug studies is increasingly raising ethical concerns. Currently, the most commonly used cell-based assays have serious predictive limitations, since the cells employed are not derived from heart cells, and may even be derived from animals rather than humans. Human cardiac-like cells are likely to give a more clinically model of drug action in people; hence there is great scientific and commercial interest in the application of human embryonic stem (hES) cells to derive cardiac-like cells for use in cellular assays and clinical applications.
Current strategies to make cardiac cells from hES produce limited numbers of the appropriate cell type, mixed in with other types of cells. Selecting the cardiac cells requires laborious mechanical procedures to enrich for the cells the researcher desires. We propose to genetically modify an hES cell line to express genes which specifically render cardiac cells fluorescent and resistant to antibiotics. These cells could then be isolated either by well-established techniques based upon separation of fluorescent cells from non-fluorescent cells, or by treatment of the cells with the antibiotic, to eliminate those cells which are not resistant to the drug (i.e., non-cardiac cells). These cells will then be tested for their reproducible performance in drug cardiotoxicity assays. The engineered cells may yield important insights into drug-induced LQTS and sudden cardiac death, and may eliminate potentially cardiotoxic drugs at an early stage in drug development, leading to more cost-effective drugs and improved health care.
Statement of Benefit to California:
Cardiac drug side effects are a major reason for compound withdrawal from development or clinical trials. The successful application of hES-derived cardiomyocytes in vitro toxicology screens will improve the safety profiles of new drugs, potentially eliminating harmful ones and avoiding errant elimination of beneficial compounds which otherwise might be dropped, with obvious health benefits to patients. Additionally, early elimination of cardiotoxic drugs from the development process reduces the financial risk to drug companies, resulting in more cost-effective development of drugs, which should be passed onto California consumers. Generation of this engineered line will yield a valuable tool for pharmaceutical development, and could yield more jobs in California’s biotechnology field. Additionally, demonstration of the utility of these cell lines in the cardiotoxicity assays may lead to the development of new products, instrumentation technologies, and other related businesses located in the State of California.
This is a proposal to select and purify cardiomyocyte subtypes (atrial, ventricular and pacemaker cells) derived from human embryonic stem (hES) cells. The investigator proposes to generate hES cell lines expressing a fluorescent marker and an antibiotic resistance gene that would allow selection of cardiomyocytes during differentiation. The proposal has three aims: (1) to produce genetically engineered cardiomyocytes by targeting the fluorescent protein and the antibiotic resistance genes to a cardiac gene locus, (2) to establish appropriate antibiotic selection conditions for enrichment of cardiomyocytes from hESCs, and (3) to use the hESC-derived cardiomyocytes for cardiotoxicity assays, exposing cells to 12 different antiarrhythmics, cardiotoxins, and drugs known to cause long QT syndrome.
Successfully purifying cardiac cell subtypes, reviewers agreed, would provide the cardiac research community with a very valuable resource. These cells generated by this project would be useful for a variety of studies, including high throughput drug screening and screening for cardiac toxicity (long QT syndrome) early during drug development. The impact of the work, if successful, would be high.
Reviewers were concerned about the project’s feasibility, mostly commenting that the research plan was not detailed enough to allow reviewers to judge its merit. Most reviewers commented that the proposal did not contain sufficient preliminary data, and that there was very little discussion of potential limitations and alternative approaches for any of the aims. For Aims 1 and 2, for instance, although reviewers agreed that the proposed genetic engineering method would probably work, they would have appreciated data to support it. The application also lacked evidence that the antibiotic treatment does not alter function or maturation of the cardiomyocytes. For Aim 3, reviewers commented that the application lacked a focused, concise discussion of the characterization of the cells, and that the rationale behind the choices of molecular markers or functional assays was weakly developed – for instance, reviewers were curious about why the cardiomyocyte markers that were chosen for qRT-PCR did not include ion channels that were being measured by electrophysiology. There were few details in the application about the electrophysiological analysis that would be performed: although the analysis includes measurement of action potential waveforms and voltage-clamp studies, reviewers remained unclear about the exact currents that would be evaluated and the rationale behind these choices. Voltage clamp records in the Preliminary Data section lack voltage protocols or descriptions of how the plots were constructed (there was little description of the plots and it was unclear whether certain plots were showing peak currents or tail currents). Considering the expertise of the collaborators, reviewers felt it was likely that these studies would be well executed, but a more scholarly development of the electrophysiological studies was required for this grant application. Overall, they commented that the characterization of the cell lines seemed unfocused and not very carefully thought-out.
The PI, who received a PhD in chemistry and completed a post-doctoral fellowship in targeted gene therapy at a top California institution, has experience in molecular biology techniques and has derived several engineered mES cell lines. The PI has a solid publication record and scientific background appropriate for this proposal, but limited managerial experience. The co-investigator has 10 years experience working with mES and hES cell lines. Collaborators at a California academic institution include experienced electrophysiologists and patch clampers (required to document cardiac purity of the cell line) with an excellent track record in cardiac electrophysiology and stem cell biology. An additional consultant on the project is an expert on directed differentiation of mesendoderm derivatives from ES lines. Other investigators seem adequately trained.
The budget is primarily dedicated to investigators’ salaries, with only 10% per year allocated for supplies. The ratio of personnel to salary is normally closer to 70:30. It is not clear that this large number of investigators is required for this project, and the supply budget seems inadequate for the number of investigators. One reviewer commented that if the characterization of the cells was more focused and hypothesis-driven, the contribution of individual investigators would be more clear. Reviewers commented that including the salary of executive personnel is not appropriate on a research grant budget sheet.
In conclusion, although reviewers were very supportive of the aims of the application and of the credentials of the research team, they did not feel that the research plan was adequately conceptualized or explained in this proposal, and they did not think that the budget was appropriate.