The therapeutic use of stem cells depends on the availability of pluripotent cells that are not limited by technical, ethical or immunological considerations. The goal of this proposal is to develop and bank safe and well-characterized patient-specific pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have the potential to develop into any cell type or tissue type in the body. Importantly, the generation of these cells does not require human embryos or human eggs. Since these cells can be derived directly from patients, they will be genetically identical to the patient, and cannot be rejected by the immune system. This concept opens the door to the generation of patient-specific stem cell lines with unlimited differentiation potential. While the current iPS cell technology enables us now to generate patient-specific stem cells, this technology has not yet been applied to derive disease-specific human stem cell lines for laboratory study. Importantly, these new cells are also not yet suitable for use in transplantation medicine. For example, the current method to make these cells uses retroviruses and genes that could generate tumors or other undesirable mutations in cells derived from iPS cells. Thus, in this proposal, we aim to improve the iPS cell reprogramming method, to make these cells safer for future use in transplant medicine. We will also generate a large number of iPS lines of different genetic or disease backgrounds, to allow us to characterize these cells for function and as targets to study new therapeutic approaches for various diseases. Lastly, we will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases.
Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have, similar to embryonic stem cells, the potential to develop into any cell type or tissue type in the body. This new technology holds great promise for patient-specific stem-cell based therapies, the production of in vitro models for human disease, and is thought to provide the opportunity to perform experiments in human cells that were not previously possible, such as screening for compounds that inhibit or reverse disease progression. The advantage of using iPS cells for transplantation medicine would be that the patient’s own cells would be reprogrammed to an embryonic stem cell state and therefore, when transplanted back into the patient, the cells would not be attacked and destroyed by the body's immune system. Importantly, these new cells are not yet suitable for use in transplantation medicine or studies of human diseases, as their derivation results in permanent genetic changes, and their differentiation potential has not been fully studied. The goal of this proposal is to develop and bank genetically unmodified and well-characterized iPS cell lines of different genetic or disease backgrounds that can be used to characterize these cells for function and as targets to study new therapeutic approaches for various human diseases. We will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases. Taken together, this would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from such research. Californians will also benefit greatly as these studies should speed the transition of iPS cells to clinical use, allowing faster development of stem cell-based therapies.
The goal of this proposal is to develop and bank safe and well-characterized patient-specific pluripotent stem cell lines. First, methods will be developed to generate induced pluripotent stem (iPS) cells by approaches that avoid the use of retroviral vectors. Second, iPS cell lines will be derived from individuals of different gender, age, and racial and ethnic origin. Additionally, new stem cell lines will be generated from individuals with certain genetic or organic disorders such as Duchenne Muscular Dystrophy, Amyotrophic Lateral Sclerosis, melanoma and other cancers. Finally, procedures will be developed for production of iPS cells under good manufacturing practices (GMP) standards. These methods and the cell lines derived in this project will be made available for the study of disease and the development of specific cellular therapies.
This is a proposal of high significance. Safe cell lines are needed for a broad range of studies and for the development of therapies, and this approach has good potential for yielding such lines. Moreover, the proposed research addresses the important goal of deriving stem cell lines from individuals of genetically diverse backgrounds. Techniques and GMP procedures developed in this project will play an important role in advancing the field toward clinical application.
The proposed research was viewed as challenging but feasible. New molecular strategies were proposed for overcoming major experimental obstacles. Appropriate procedures for the analysis of new cell lines were presented. While somewhat meager, preliminary data demonstrate the principal investigator (PI)’s proficiency with iPS cell derivation. The PI is highly experienced, productive, and qualified to carry out the proposed research. Co-investigators have appropriate and complementary expertise for facilitating research progress. The institutional environment provides an excellent setting for performing the proposed research.
Reviewers had some concern that the proposal was overly ambitious. Nevertheless, the potential benefits and value of the experimental advances were viewed as outweighing the risks. The experience and proficiency of the research team heightened reviewers’ confidence that the project will be successful.
The application is highly responsive to the RFA. Clinical-grade and disease-specific stem cell lines will be created, characterized, and shared. Both the methods and specific cell lines developed in this project will contribute significantly to advancing regenerative medicine.
Programmatic Discussion: A motion was made to recommend that this application be moved to Tier 1 - Recommended for Funding. Reviewers felt that concerns about the likelihood for success were outweighed by the importance and potential benefits of the project. The high quality of the PI and the introduction of new technologies for making iPS cells were viewed as particular strengths of the proposal. The motion to move this application to Tier 1 carried.
This proposal calls for the generation of clinical grade iPS cell lines useful for both patient-specific therapy and for the study of disease-specific research. The current published approaches using retroviral delivery of “pluripotency” factors are problematic, both because some of the factors themselves could be oncogenic and the concern with random integration of the viral vectors into the host genome. To address these concerns, the investigators are proposing to use relatively novel transient expression/delivery of the required reprogramming factors, and to do this in a manner that is ultimately compliant with GMP manufacturing so that the newly derived lines could be qualified for therapeutic use. Furthermore, the new iPS lines will have genetically diverse backgrounds.
Reviewer One Comments
This proposal targets the development and banking of safe and well-characterized patient-specific pluripotent stem cell lines for use in the study of human disease. This proposal has a high degree of significance, as these safe cell lines are needed for a broad range of studies.
There are three specific aims in this proposal. The first targets molecular strategies to develop safe iPS cells. The second aim is to generate a bank of iPS cell lines from people of different sex, age, and diverse population and disease backgrounds. The third specific aim is to establish standard operating procedures of cGMP derivation, expansion and banking of human iPS cells.
The PI has a good track record of publications and experience generating iPS. Collaborators include Drs. Plath and Lowry (5% effort each, methods to develop iPS cells), Dr. Wyatt (10% effort, behavioral scientist identifying diverse population in the LA area), and Dr. Quan (10% effort, FDA regulations and GMP procedures).
The environment at UCLA is an excellent one for the successful accomplishment of the goals of this proposal. Resources include a GMP facility, a CIRM sponsored shared research laboratory, and the NUDE/SCID Mouse Shared Resource Facility.
Responsiveness to RFA:
This proposal is adequately responsive to the RFA.
Reviewer Two Comments
This proposal calls for the generation of clinical grade iPS cell lines, useful for both patient-specific therapy, and for the study of disease-specific research. The current published approach using retroviral delivery of “pluripotency” factors is problematic, both because some of the factors themselves could be oncogenic, and the concern with random integration of the viral vectors into the host genome. To address these concerns, the investigators are proposing to use relatively novel transient expression/delivery of the required reprogramming factors, and to do this in a manner that is ultimately compliant with GMP manufacturing so that the newly derived lines could be qualified for therapeutic use. Furthermore, the new iPS lines will have genetically diverse backgrounds.
Design and Feasibility
This proposal calls for using a variety of new molecular strategies to reprogram cells, attempting to avoid retrovirus mediated delivery. The investigators show preliminary data that they have had some success with the established methods for generating iPS. They plan to start with adult skin fibroblasts as original source material (obtained from commercial suppliers, this would be problematic for subsequent GMP qualification)
One innovative aspect is to combine three reprogramming factors into one vector to ensure equivalent expression in each cell. It is an interesting hypothesis that similar levels of expression of the factors within individual cells will lead to more efficient generation of iPS, but it is clearly unknown whether this will actually prove to be the case. They are proposing to start with Lenti vector, but will switch to Adeno (non-integrating). This should lead to gradual dilution/loss of the vector during the derivation/expansion phase. The expectation is that transient expression will be sufficient, and there is some preliminary data using PCR that shows silencing of vectors after reprogramming is completed. They are also proposing to evaluate other options to introduce the reprogramming factors, such as nucleofection or transduction of whole proteins (with HIV-TAT or electroporation). Finally, they also mention plans to control the duration of expression by the Tet-activator system. All of these options may or may not work (high risk/high reward).
Standard approaches for the characterization assays of the iPS for pluripotency and self-renewal are included in the proposal. They plan to compare these with established hESC. They will then apply the optimal reprogramming methods to create a larger bank of iPS lines for research and subsequent clinical therapy. They have access to a diverse population of individuals through the UCLA clinics, including several possible disease models:
1. Muscular Dystrophy
They also propose to develop these reprogramming methods for cGMP-compliant production for therapeutics. This will be developed as a core facility, no direct clinical studies are proposed. Several aspects are mentioned:
- Derivation protocols that are feeder cell free (use of commercial media). It was not clear what the GMP-compliant status of these commercial media was?
- Expansion - minimal details were provided on exactly how this would be accomplished, and to what extent the proposed methods would be GMP compliant.
- Banking - again, little detail provided to indicate that the investigators understood the issues associated with qualification of master and working cell banks.
The investigators do have some experience with FDA regulated gene therapy clinical studies, and there is an established GMP-compliant facility in place at UCLA.
The PI is well-funded:
1. Pathogenesis of HIV
2. HIV latency
3. CIRM grant hESC strategies to target HIV
4. CFAR grant for HIV
5. CIRM grant for multilineage hESC differentiation into hematopoietic lineage
6. CIRM Generation of pluripotent hESC (GMP production)
7. Core director for mouse shared research facility
This is an overly ambitious proposal; it is difficult to see how the investigators will be able to accomplish all of their specific aims in the short timeframe. As such, the PI is only listed on the RFA at 10%, with co-investigators at 5%, 5%, 10%. All other key personnel are not yet in place (to be determined), and no consultants are listed.
Responsiveness to RFA:
This proposal is clearly very responsiveness to the RFA. The investigators are proposing to generate both clinical-grade as well as disease-specific iPS, using some innovative approaches for derivation.
Reviewer Three Comments
Significance and Innovation: High.
The author proposes to develop technology to generate safe iPS cell lines. They will stress the development of technologies that could only transiently express the reprogramming genes to avoid retroviral insertional mutagenesis. They propose to help develop standard protocols to evaluate pluripotency and self renewal of the newly created lines. In aim 2 they propose to generate iPS lines from people with diverse background and diseases and make the cell lines available to researchers. In aim 3 they propose to establish SOP and cGMP procedures for derivation and expansion and banking of the lines created.
Design and Feasibility of the Research Plan:
First, the authors have successfully developed methods to create iPS lines from both mouse and human cells and have characterized the cells for pluripotent characteristics. The lines were created through traditional methods, yet these results give the application credibility. The authors have proposed several strategies to produce safe iPS cells from including the reprogramming genes on one lentiviral vector to reduce the number of integration sites. Alternative strategies will try to avoid viral integration through the use adenoviral vectors with or without selectable markers so that the episomal transgenes will be lost through subsequent replication. Unintended integration will be assessed by FISH analysis for the transgenes. Finally they suggest they could transduce the 4 factors and proteins with a short TAT peptide of HIV that enables proteins to cross plasma membranes as is done with Cre-recombinase.
Resultant cells will be tested for pluripotency via traditional mechanisms and differentiation potential (hematopoietic, myeloid and erythroid). They will try to correlate gene expression patterns with differentiation potential.
The plan to generate iPS lines from a diverse population of individuals with and without background diseases is considered useful. Even traditional iPS cell line generation would likely be sufficient to make useful cell lines for research purposes. The team suggests that up to 30 individuals of diverse ethnic background will be used as well as people with diseases such as muscular dystrophy ALS, melanoma and other cancers.
The authors also propose to generate iPS lines with feeder free techniques. While most of the experiments proposed in the application are highly speculative, the team has considerable experience and success with the iPS technology and at least some of the proposed methods will likely work.
In aim 3 they propose to establish cGMP procedures for cell line generation. The group has access to the clean rooms and the GMP level facilities to accomplish the aim if aims 1 and 2 are successful. The proposal suggests that technique development and refinement would not be done under GMP conditions, but that useful protocols once established would be adapted to GMP standards. The PI, Dr. Zack has experience with GMP level clinical research with hematopoietic stem cell gene therapy trials. The team also includes Dr. Quan, director of regulatory compliance for other research protocols.
PI and Personnel
Dr. Jerome Zack is Professor in the Department of Hematology and Oncology, UCLA School of Medicine. He is director of the UCLA Broad center human embryonic stem cell bank and is associate director of the Eli and Edyth Broad center of regenerative medicine and stem cell research at UCLA. He has over 125 publications and considerable research support. He is highly qualified to lead this team. He has significant help from other investigators in particular Kathrin Plath and William Lowry for the generation of iPS cells and Dr. Quan for regulatory affairs.
Overall evaluation: Although the application is highly speculative and requires significant development of technology, the team assembled has considerable experience in the field and has demonstrated proficiency with the iPS technology GMP level investigations and other techniques needed to successfully complete the proposed research.
Responsiveness to RFA:
Lines will be characterized and shared.