Funding opportunities

Novel non-integrating stem cell-derived rAAV vectors for safe and efficient iPSC induction

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01887
Funds requested: 
$1 808 881
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
The ability to safely and efficiently generate induced pluripotent stem cells from normal cells of the body without the permanent introduction of foreign DNA holds tremendous promise for the production of patient-specific stem cells for genetic correction of inherited diseases, regenerative medicine and transplantation. These primitive stem cells, like embryonic stem cells, have the capacity to develop into any type of cell. Yet they can be derived from any individual’s cells after birth. The availability of a person’s own stem cells would overcome enormous public health and economic barriers by removing the need for tissue matching and maintenance of tissue banks and registries of tissue type-matched donors for transplantations. Moreover, availability of primitive stem cells of specific disease origin has the potential to play a key role in studying human diseases. The biggest hurdle with this promising technology lies in the permanent introduction of foreign DNA during the derivation of these stem cells. This carries a major risk of inducing mutations that may lead to cancer and other genetic problems, thus precluding their use for human therapy. The low efficiency of creating stem cells by other safer methods also precludes their use in large scale. Here we propose to use blood cell-derived isolates of a safe and commonly occurring virus that has never been associated with any human disease known as adeno-associated virus. A family of this virus was recently cloned in our laboratory, and was found to have very high gene delivery efficiencies. Gene delivery vectors designed from this virus do not become a permanent part of the cell’s DNA and they are lost within a few days of infection. Thus they are ideal for the temporary expression of foreign genes without inducing permanent genetic changes. Here we propose to test the generation of induced pluripotent stem cells using gene delivery with this unique family of viruses. We propose to test their ability to deliver reprogramming genes to blood cells for the purpose of generating induced pluripotent stem cells without changing the cell’s DNA. This project is based upon a body of convincing experimental data that strongly suggests that this approach will be successful. When completed, this project would address the most significant challenge to date in the generation of induced pluripotent stem cells and will provide a means of safely and efficiently deriving them from individual patients. Stem cells derived by this approach will not contain any foreign genetic material and therefore will be safe for widespread clinical use. These stem cells may then be used for therapeutic transplantation and regenerative medicine. The significant strength of this project lies in its ability to efficiently and safely generate induced pluripotent stem cells without changing the cell’s genetic material, thus addressing the most significant roadblock to progress in this exciting field.
Statement of Benefit to California: 
The ability to safely and efficiently generate induced pluripotent stem cells from normal cells of the body holds tremendous promise for the production of patient-specific stem cells for genetic correction of inherited diseases, regenerative medicine and transplantation. These primitive stem cells, are similar to embryonic stem cells and have the capacity to develop into any type of cell. Yet they can be derived from any individual’s cells after birth by expression of a certain set of reprogramming genes. The biggest hurdle with this promising technology lies in the permanent introduction of foreign DNA during the derivation of these stem cells. This introduces a major risk of inducing mutations that may lead to cancer and other genetic problems, thus precluding their widespread use for human therapy. The low efficiency of creating stem cells by other safer methods is also problematic. Here we propose to use blood cell-derived isolates of a safe and commonly occurring virus that has never been associated with any human disease known as adeno-associated virus. Gene delivery vectors designed from this virus do not become a permanent part of the cell’s DNA and are lost within days of infection. Thus they are ideal for the temporary expression of foreign genes without inducing permanent genetic changes. Here we propose to test the generation of induced pluripotent stem cells using gene delivery with this unique family of viruses. We propose to test their ability to deliver reprogramming genes to blood cells for the purpose of generating induced pluripotent stem cells without changing the cell’s DNA. When completed, the research proposed here will develop a way to safely and efficiently generate induced pluripotent stem cells from normal cells of the body without the permanent introduction of foreign DNA. These primitive stem cells have the capacity to develop into any cell type of the body. This project will benefit state of California and its citizens by making it possible to safely and efficiently derive stem cells from any individuals who need them for either transplantation or regenerative medicine, for example, in the treatment of neurodegenerative diseases or for blood cell transplantation. When completed, our approach to safely generating stem cells would lead to the widespread use of induced pluripotent stem cells. This would overcome enormous public health and economic barriers, and remove the need for tissue matching, maintenance of tissue banks and registries of tissue type-matched donors for transplantations. Deriving induced pluripotent stem cells of specific disease origin would also have the potential to play an important role in studying human disease processes. Thus, the major strength of this project lies in its ability to efficiently and safely generate stem cells without changing the cell’s genetic material, thus addressing the most significant roadblock to progress in this exciting field.
Review Summary: 
The goal of this proposal is to develop a safer, more efficient means for deriving induced pluripotent stem cells (iPSC) using non-pathogenic, non-integrating adeno-associated virus (AAV) vectors. Currently, the lack of efficient methods for deriving iPSCs via non-viral, or non-gene modifying approaches is a major obstacle in the field, thus precluding consideration of these cells for clinical applications. To address this translational bottleneck, a series of three aims has been proposed. First, the applicant will evaluate high efficiency, non-integrating recombinant AAV (rAAV) for delivery of reprogramming genes to somatic cells for iPSC generation. Aim 2 will focus on confirmation that rAAV transduction does not result in genomic integration of viral sequences. For the final Aim, the applicant proposes to fully characterize iPSC generated by rAAV transduction and test their pluripotentiality and capacity for differentiation. In general, reviewers appreciated the rationale for using rAAV to develop a “hit and run” strategy for transient expression of reprogramming factors with minimal risk of genetic modification. They agreed that if successful, this project could have a major impact on the field. However, while they appreciated novel attributes of the proposed AAV subtype, most reviewers felt that the overall project was only moderately innovative. Several vectors (targeted and non-integrating, including AAV) have already been developed for iPSC generation. Furthermore, several promising nonviral methods have been recently described that ameliorate the risk of insertional mutagenesis. Reviewers discussed a number of strengths and weaknesses in the research plan. While the overall approach appeared straightforward and logical, there was no preliminary evidence indicating that the levels of expression afforded by transient presence of rAAV would be sufficient to elicit cell reprogramming. Furthermore, there were no data to establish the applicant’s technical capabilities with respect to culture and derivation of pluripotent cells. Potential pitfalls and alternative solutions were also not adequately addressed. In contrast, reviewers were enthusiastic about the rAAV serotype under investigation, noting that CD34+ cells represent an interesting and attractive source for manipulation. Despite the favorable properties of rAAV, however, reviewers were uncertain whether it would be possible to prove that all virus is indeed cleared, or that integration does not occur at extremely low levels. Given these concerns, reviewers suggested that experiments to track or sort infected cells should have been included. Finally, reviewers noted that much of Aim 1 appeared redundant with preliminary studies, and Aim 2 represented only a slight extension of Aim 1. Due to these partially met objectives, the rapid kinetics of rAAV infection, and the prior development of needed tools, reviewers felt that the studies that would be undertaken to complete the work lacked ambition and were perhaps insufficient for a grant award. Reviewers described the principal investigator (PI) as an established, well published expert in the development of AAV vectors for stem cell gene transfer. Team members were also found to be qualified, although lacking in demonstrable expertise with human iPSC generation. Several reviewers felt that the project was overstaffed, as the lean experimental objectives could likely be carried with half as many individuals and in significantly less time than allotted. The PI’s institutional support and environment were considered excellent. Overall, reviewers appreciated some novel aspects of the proposed tool and considered the potential for impact to be high. Nonetheless, their enthusiasm was diminished by uncertain feasibility, an overgenerous timeline, and a recent spate of alternative approaches for safer and more efficient iPSC production.
Programmatic review: 
  • This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was suggested, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.
Conflicts: 

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