Optimization Of AAV/Cas9 Genome Editing System For Gene Therapy

AAV vector has become one of the most promising gene delivery vectors in gene therapy because of its non-pathogenicity,broad tropism and persistent transgene expression.The CRISPR/Cas system is a well-developed genome editing system with accurate genome editing efficiency.The AAV-Cas9 in vivo genome editing system has great potential in the field of genetic research and genetic medicine.However,there are still some problems to be solved.Firstly,preexisting AAV neutralizing antibodies greatly impede the gene transduction of AAV vector.Test of AAV neutralizing antibody is a necessary step in clinical trials.Conventional detection method of AAV neutralizing antibody is based on the transfection of cells.Briefly,the serum sample is gradiently diluted and mixed with the AAV vector carrying reporter gene and the resulting mixture is used to transfect corresponding cells.Serum dilution folds that result in 50% decrease in reporter signal is defined as levels of neutralizing antibodies in serum samples.Here we designed a new neutralizing antibody detection method based on theoretical hypothesis that AAV transfection efficiency is dependent on AAV-cell binding.First,we demonstrated that the number of AAV vectors bound to cells is positively correlated with the number of added AAV vectors,regardless of transfection efficiency.Second,the number of AAV vecots bound by the neutralizing antibody can be calculated by quantifying the number of cell-bound AAV vectors by q PCR,thereby estimating the level of neutralizing antibodies in the sample.Finally,the new method was used to detect AAV neutralizing antibody in clinical samples and generated results in aggrement with those from conventional method.The new detection method is simple and fast,and makes it possible to develop a standardized process for AAV neutralizing antibody detection.Secondly,the AAV vector has limited load capacity and the DNA elements needed for genome editing must be distributed in two AAV vectors for in vivo administration,which has a certain influence on the efficiency of gene transduction and gene editing.Moreover,AAV-mediated DNA delivery results in the sustained expression of Cas9 gene in cells,which not only increases the likelihood of off-targeting of the editing system,but also increases the risk of immunotoxicity of Cas9 in cells.We pioneered attempt to incorporate Cas9 protein into AAV vector and build an AAV-Cas9 all-in-one genome editing system which can both free the load capacity of the AAV vector and reduce the genotoxicity and immunotoxicity of Cas9 in cells.First,in order to detect and evaluate the activity of gene editing,we optimized the fluorescence defect repair system to detect gene editing activity.Secondly,based on the understanding of the structure and modification of AAV capsid protein,we designed a Sp Cas9-VP2 fusion strategy and successfully constructed the fusion protein with DNA recognition and cleavage function.Finally,the obtained fusion protein gene was transfected into HEK293 cells with other AAV replication-related plasmids to produce AAV packaging products.Liver cancer derived cells transfected with AAV/Cas9 fusion vector products showed positive in gene editing activity.This study provided a feasible idea for construction of AAV-Cas9 all-in-one gene editing tool.In summary,we proposed and explored solutions to two clinical application problems of the AAV-Cas9 in vivo genome editing system.This study established a new method for the detection of AAV neutralizing antibodies and verified the new idea for construction of AAV-Cas9 all-in-one genome editing tool,providing experiment basis for optimization of genome editing mediated by AAV vector delivery.