As a first step for the application of the CRiSPR/Cas9 (Clustered Regularly Interspaced
Short Palindromic Repeats) gene editing system as a therapeutic approach for FOXG1 mutated patients, we have studied the best strategy for obtaining an efficient and specific infection in iPSCs derived from Rett patients. We have searched for a strategy that could also be amenable to transfer to patients’ treatment for future clinical trials with minimal modifications. For this purpose, we have analyzed available literature data on the tropism of the different AAV (Adeno-Associated Viruses) serotypes and we have selected AAV2, which, while keeping absence of immunogenicity, has been proven to be the best serotype to infect the cell lines that represent our targets and to be able to pass through the blood-brain barrier. As an anticipation of the first 6 months of the project, we already worked on the
construction of the CRiSPR/Cas9 gene editing vectors. Specifically, our strategy is based on the construction of two vectors for co-infection for contemporary delivery into the cells of the two components of the CRiSPR/Cas9 system. The first vector brings the Cas9 (the genetic scissors), with two flanking target regions for auto-cleaving of the Cas9 itself. This strategy avoids the prolonged expression of Cas9, thus preventing multiple cuttings in un-wanted genomic regions and increasing the safety of the system. The second vector is constructed in order to bring the Donor DNA with the wild type sequence that will replace the mutated one; this vector also brings the mCherry reporter system, that will allow to visualize infected cells since they will become fluorescent, and the sgRNA, under the control of the U6 Human Promoter, that is necessary to properly direct the Cas9 on the specific nucleotides that we want to correct. These experiments are ongoing for correction the following 2 specific mutations in FOXG1 gene, - c.640Gdup - c.688C>T, for which iPS cells are already available. Once completed, the two vectors will be encapsulated inside AAV2 viruses to test the system directly on the iPS cells.
12 months agoWe have collected more then 79’000 euro and a commitment for the remaining of the funds from two Swiss Foundations who have generously provided the bulk of the amount needed for this research.
Thank you to every person who has contributed to this campaign. We have started the project and hope to complete by July 2019. We will keep you updated through curefoxg1.com website.