Adaptive Immunity Of AAVs And Cas Proteins In Rhesus And Cynomolgus Monkeys

Gene therapy provides a new alternative for some genetic diseases.AAV is the most commonly used viral vector in gene therapy,and has made breakthroughs in gene therapy for some diseases for future clinical trials.With the rapid development of gene editing technology based on the CRIPSR system,the genetic modification of mammalian cells has become efficient and convenient.The gene therapy based on AAV-CRISPR makes it possible to cure most genetic diseases,while the adaptive immunity of AAVs and CRISPR-associated proteins(Cas),as an exotic substances,may greatly weaken the efficacy of gene therapy,even induce some unknown risks.Therefore,to learn the adaptive immunity of AAVs and Cas proteins in vivo is very important as a practical reference for the safety and effectiveness of gene therapy.Non-human primates have highly similarity to humans in genome homology,evolutionary relationship and physiological characteristics,so non-human primate models can simulate humans well in biomedical research.In order to analyze the preexisting adaptive immunity against AAVs and Cas proteins in primates,the following experiments have been carried out and the main results are listed below:Part 1: Prokaryotic expression and protein purification of Cas proteins.By designing and constructing prokaryotic expression vectors of SaCas9,SpCas9,AsCas12a,and LbCas12a,IPTG was used to induce the production of recombinant proteins,and these four proteins were isolated and purified by immobilized metal ion affinity chromatography.Part 2: Screening for pre-existing AAVs and Cas protein antibodies in the sera of three animals.Specific antibodies to AAVs and Cas proteins can be detected by Western Blot and ELISA indirect methods.We used recombinant AAVs or Cas proteins as antigens to detect pre-existing specific IgG antibodies in the serum,and conducted extensive tests on monkey,dog and mouse populations.The ELISA results for AAVs antibody in the monkey population showed that the AAV8 and AAV9 antibodies were widely positive.At the same time,Western Blot and ELISA results showed that IgG antibodies against four Cas protein homologs of SaCas9,SpCas9,AsCas12a,and LbCas12a were also prevalent in sera of monkeys,mice,and dogs.Part 3: Immunization monitoring of AAVs and Cas proteins injection in mice and monkeys.The pre-existing antibodies in the serum can only reflect the strength of the antibody at that time,and there is no way to further determine the strength of adaptive immunity.Intravenous injection of recombinant AAV and Cas protein can stimulate a strong adaptive immune response,which can effectively simulate the immune response of AAV-CRISPR in vivo,so as to know the adaptive immune of AAV-CRISPR gene therapy in vivo.We continually monitored the humoral and cellular immunity by injecting low-dose recombinant AAV9 and SpCas9 protein into mice and monkeys.The results showed that recombinant AAV9 and SpCas9 protein can effectively trigger secondary immunity.At the same time,we monitored the cellular immune response of the low-dose AAV9 and SpCas9 protein by flow cytometry within 24 hours.The results showed that the low-dose AAV9 virus and SpCas9 protein did not change significantly during the monitoring period.Conclusion: It was found that pre-existing adaptive immunity against AAVs and Cas proteins prevalent in monkeys,which provides a good reference for the formulation and implementation of gene therapy based on AAV-CRISPR.