A brand new paper by the Serrano group explores in vivo reprogramming in mice with AAV. Manuel talked about it at the Glenn Symposium, but I would like to cover that topic in more detail.
The pilot experiment was done in vitro in MEF (mouse embryonic fibroblast) cells. The reprogramming factors were delivered by co-transducing the cells with four individual AAV vectors containing O/K/S/M. The group developed a new vector configuration, the (sc)AAV vector (self-complementary). This resulted in a much more efficient transduction with the expression of the factors in over 80% of all cells. Formation of iPSCs was assessed by immunofluorescence and RT-PCR as well as by injection of iPSCs into mice with consequent occurrence of teratomas.
Then the reprogramming was performed in vivo with the delivery of all four factors packaged together in AAV8. The results appeared to be dependent on the AAV dosage. Moreover, the reprogramming efficiently occurred even without the c-Myc oncogene. The reprogramming efficiency was 0.1% and 0.2% for the low and high dose respectively which is much higher than in previous studies: 0.001-0.091%. The mice after the viral injection developed teratomas from which iPSCs were derived that were checked for pluripotency by again the injection into nude mice. Interestingly, different tissues acquired structural abnormalities with very different speeds. The information about the pathologies found after two and four weeks post-injection can be found in the supplementary material.
Most importantly, the authors stated that the choice of the delivery system was based on the safety of AAVs that was proved by over 160 clinical trials. This work studied if there were viral integrations in the genome and if there were where they occurred. AAVs are believed to integrate into the genome at a very low rate. Surprisingly, here integrations were found in three quarters of iPSC clones generated in vitro and in all in vivo iPSC clones with an average of 2-40 insertions per clone. Integrations were found both in fully and partially reprogrammed clones that implies that they happened early during dedifferentiation. There were no specific regions with higher insertions. Half of the insertions were within genes, but only 10 out of 180 insertions were in exons, and two or three of them were in genes associated with cancer depending on the cancer gene database.
The authors believe that integrations of AAV into the genome are required for the reprogramming process. AAV vector insertion likely happens due to the remodeling of chromatin that occurs during cellular dedifferentiation or rapid cell division and makes chromatin open and accessible. They substantiate that with multiple references. Since the iPS cell can further differentiate into many cell types, this system can help study the consequence of insertions into different regions. More generally, AAV-mediated iPSs could be a great model to investigate the biology of AAV for further gene therapy advancement.
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