Statement of Benefit to California:
Year 1The original goals in the CIRM application were to use molecular DNA shuffling to make an AAV capsid library and then use the library to select for AAV vectors that would efficiently transduce human embryonic stem cells. We spent a long time preparing the capsid library and proving that our library had diverse sequences. In the end, we made two libraries each with about 100,000 variant capsids. We did our first set of screens on cultured cell lines and pulled out several novel AAV capsids. We vectorized them and showed they were much more efficient at transducing cells in culture than any other serotype described to date. There is a large amount of data describing the experiments in detail. These studies were published in a very long article published in the Journal of Virology in mid 2008 (Grimm et al., 2008.. In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies
Interbreeding and Retargeting of Adeno-Associated Viruses . J. Virol. 82:5887-5911
We had started using the capsid library in human embryonic stem cells as described. We had some technical problems with titering the right amount of human adenovirus needed as a helper virus in the designed screen. Unfortunately, time ran out and the grant ended and a new proposal to continue the work was not selected for review by CIRM. Nevertheless, since we believe producing the reagents we proposed to make for human ES stem cells are important for the research community and now that we have two very robust and diverse libraries, we are going to attempt another round of capsid screening. If we obtain useful serotypes for human ES cell transduction, we will make them available to the research community.
We should point out that we have recently produced DNA minicircle vectors, which are episomal plasmids that lack all bacterial plasmid backbone DNA. These minicircles have been used in collaboration with Joesph Wu and Michael Longaker to convert human adipose derived cells into iPS cells with 10 to 20 times greater efficiency than plasmid DNA. Moreover, these vectors are only transiently present in the iPS cells establishing that they do not permanently alter the genetic makeup of the cells. This work while not funded by the CIRM may turn out to be a more valuable approach for reprogramming all types of stem cells. If it were not for CIRM funding, this collaboration may not have occurred. The CIRM funding brought my group together with Joseph WU and Michael Longaker.