Funding opportunities

Novel vectors for gene transfer into human ES cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00236
Principle Investigator: 
Institution: 
Funds requested: 
$640 642
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Human embryonic stem cells have a great potential for medical therapeutics. However, the genes required for altering the fate of these cells to differentiate into a particular tissue or cell type is not well understood. The ability to efficiently transfer genes or silence genes in ES cells would be of great benefit for two reasons: 1) A combination of gene therapy and ES stem cells will likely broaden the therapeutic potential of these cells, and 2) the ability to alter gene expression will provide important tools for unraveling the genetic programs required for targeted differentiation into a specific tissue or cell-type. Viral gene transfer vectors are derived from viruses. The viral genes are removed and replaced with a therapeutic gene or a gene under biological study. Thus, the viral shell is used to transfer the desired gene into cells. Although there has been some success, current gene transfer vectors do not work efficiently in ES cells. To make this new gene transfer vector, we will use the adenoassociated virus (AAV). This virus is not associated with any known disease and has been used in a number of human clinical trials. Another advantage of vectors based on these viruses is that they can be used to overproduce a gene product, turn off a gene product, and even target a mutation in a specified gene. We plan to use a recent strategy developed in our laboratory to select for AAV viruses that are very efficient at infecting human ES cells. Once these viruses are identified, as a proof-of-concept, we will make gene transfer vectors and test them in new human ES cells for their ability to over-express and turn off genes that we believe are important in early human ES cell differentiation.
Statement of Benefit to California: 
California is leading the way in stem cell research. The ability to transfer nucleic acids into stem cells will open up an additional therapeutic window as well as allow important tools for helping define the genetic programs required for stem cell differentiation into a myriad of different cell types. The intellectual property surrounding the vectors that we have selected and characterized by the same approach for liver transduction have been protected by a patent application submitted on behalf of {REDACTED}. Like all of our reagents and discoveries, the ES cell specific gene transfer vectors developed in our laboratory will be made available to all academic researchers around the world. We will offer these vectors to corporate entities per the rules and regulations dictated by the California State Stem Cell Institute and {REDACTED}. We should note that even prior to publication, several vectors isolated and characterized for liver gene transfer by our laboratory have been sent to commercial entities for evaluation and possible use in both research and clinical trial development. We believe that our technology will have wide-spread use by stem cell researchers in California as well as the rest of the world.
Review Summary: 
SYNOPSIS: In this proposal, the investigator wishes to generate AAV vectors that will transduce hESCs at high frequency. Such vectors will be constructed following screening of AAV capsid libraries generated by DNA shuffling for those viruses that most efficiently infect hESC lines. The vectors will engineered to express shRNA and the efficency of shRNA inhibition of gene expression in hESC determined. AAV vectors that allow for permanent genetic modification of hESC will be generated using a DNA transposon system. Proof of concept experiments will then be performed in hESC by knocking down and overexpressing genes known to be important during endoderm formation. The H9 cell line will be used in initial vector derivation studies, other hESC lines will be used subsequently. INNOVATION AND SIGNIFICANCE: There is a significant need for efficient methods for genetic manipulation of hESCs, both short term and long term as there are currently no efficient methods to genetically modify hES cells. Allthough some progress has been made using retroviral vectors, silencing and low frequencey of transduction remain a problem. Moreover all these methods result in stable integration of the donor DNA in the host cell. Plasmid transfection is not very efficient either. AAV vectors, with the correct capsid can transduce close to 100% of cells. A single effective method to genetically modify hESCs would have very significant implications. STRENGTHS: This is a proposal from a highly accomplished PI who is a leader in the AAV vector field with extensive experience in AAV vector development, in collaboration with investigators with extensive experience in hESC culture and evaluation. The studies proposed are well thought through. One reviewer believed it highly likely that good methods will be developed for high efficiency short term and long term genetic modification of hESCs. Another reviewer was less sanguine but had a high degree of confidence that valuable insights into possibilities of hESC genetic transduction will be obtained. This reviewer considered it somewhat questionable if AAV vectors will be effective in the hESC system but noted that the expertise of the PI generates confidence that this issue will be answered one way or another. WEAKNESSES: It is somehwat unfortunate that the methods to be developed will not be compared with other methods currently used for genetic modification of ESCs (transfection or transduction via lentiviral vectors), as this would allow the investigators to determine whether the AAV method is definitely better than established techniques. DISCUSSION: Successful generation of AAV vectors that efficiently transfect hESC is necessary for the rest of the proposed research. There was discussion about the issue of silencing which could occur with the proposed integrating vector system.
Conflicts: 

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