Statement of Benefit to California:
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.