Pre-clinical Research On The Treatment Of ? Thalassemia By Combining CRISPR/Cas9 With RAAV6 Vector

Beta thalassemia is hereditary hemolytic anemia caused by mutation of the HBB gene that leads to the reduction or deletion of?-globin chain synthesis.Thousands of beta-thalassemia carriers account for about 1.5%of the global pop?lation.Beta thalassemia is also the deadliest and disabling blood genetic disease with the highest incidence and the largest impact in southern China.The thalassaemia gene carrying rate in Guangdong is as high as 16.83%,the incidence is about 7.8%,and the severe mortality rate is about 3%.According to statistics,4,000 children with severe?-thalassemia are born in Guangdong each year,which places a heavy burden on families and society.At present,the only cure for?-thalassemia is allogeneic hematopoietic stem cell transplantation.However,fewer patients can benefit from it due to low MHC matching rate,pretreatment toxicity,and graft-versus-host disease.Gene therapy based on the patient's autologous hematopoietic stem cells has become a new approach to cure?-thalassemia.Among them,some clinical trials have been carried out for lentivirus-based gene therapy.However,due to the random integration of lentivirus,its clinical safety needs long-term follow-up observation.And gene editing mediated gene therapy has become a hotspot in the treatment of beta-thalassemia.Using gene-editing tools thro?gh the homologous recombination repair pathway can accurately repair the?thalassemia mutation site,and return the repaired autologous hematopoietic stem cells to patients is expected to completely cure thalassemia.CRISPR/Cas9 gene-editing technology has been widely used because of its simple design,operation and low price since 2012.Its RNP form has been widely used due to high gene editing efficiency,no requirements for codon optimization,promoter selection,low off-target effects,low toxicity,and immune response.Homologous recombination repair templates mainly include single-strand oligodeoxynucleotides(ss ODNs)and virus-mediated targeted modification vectors.The donor sequence of ss ODNs does not exceed 200nt,which is mainly used for the repair of specific mutation sites,we plan to establish a universal gene targeting repair scheme for common types of beta-thalassemia mutations,so we selected a virus-mediated targeted modification vector as a template.Recombinant adeno-associated virus(r AAV)is currently recognized as the safest clinical-grade gene therapy vector and has a 4.7 kb donor sequence packaging capacity.Among them,ser?m type 6 has the highest efficiency for hematopoietic stem cell infection.Therefore,we plan to use the RNP form of CRISPR/Cas9 and r AAV6 to establish a?thalassemia efficient universal gene targeting repair system,and we hope to lay a theoretical foundation for clinical treatment mediated by gene-editing technology thro?gh verifying the feasibility and safety of the scheme.Part 1.Establishment of a universal and highly efficient gene editing and repair system of HBB mutation site using cord blood-derived HSPCsObjective:To establish an efficient universal gene editing and repair system of HBB mutation site using cord blood-derived HSPCs,which co?ld construct a foundation for subsequent functional research and repair of disease-derived HSPCs.Methods:Using CCTop online design platform to design 5 sg RNAs for intron 2of HBB gene,and using Snap Gene software to design in vitro repair template r AAV6.PX458-sg RNA plasmids were constructed by vector construction technology.T7E1assay and TIDE platform were used to analyze the indel efficiency of sg RNA.Transfection of RNP complex with Neon transfection system,and transduction of r AAV6 at the same time.To obtain an efficient gene editing and repair system by optimizing the molar ratio of cas9 protein/sg RNA,electroporation parameters,r AAV6 MOI,etc.Methylcell?lose monoclonal experiments and in-out PCR technology was used to verify the genotype and ratio of HSPCs after repair.Res?lts:We successf?lly established a highly efficient universal gene editing repair system of the HBB mutation site,the indel efficiency can reach 80-90%,and the efficiency of homologous recombination repair can reach an average of 12%.The optimized parameters of the gene-editing repair system are as follows:molar ratio of cas9/sg RNA is 1:2.5;electroporation parameters are 1600V,10ms,3 p?lses;using chemically modified sg RNA;r AAV6 MOI is 10⁴.61%of cord blood-HSPCs after gene targeting are single allele repair,and 35%are double allele repair.Conclusion:We have successf?lly established an efficient universal HBB mutation site gene editing repair system by optimizing various transduction parameters,which can be used for the subsequent research on the functional and repair of HSPCs.Part 2.Verification of function and genomic off-target effects after repair of cord blood-derived HSPCsObjective:Analyzing the function and the whole-genome off-target effects of cord blood-HSPCs after gene targeting.Methods:Using the CFU assay to show the forming ability of lineage-restricted progenitors of cord blood-HSPCs after gene targeting.The directional erythroid differentiation experiment was used to detect the erythroid differentiation efficiency of HSPCs after targeting.Giemsa staining was used to detect the morphological changes of HSPCs after targeting.Mouse transplantation experiments were used to analyze the long-term reconstruction capacity of HSPCs in vivo after targeting,whole-genome high-thro?ghput sequencing was used to analyze the off-target effects of gene editing repair systems.Res?lts:The res?lts of the CFU assay showed that there was no significant difference in the formation ability of m?ltilineage progenitor cells before and after targeting.The res?lts of the directional erythroid differentiation experiment showed that there was no significant difference in the efficiency of the directional differentiation of HSPCs before and after targeting.Mouse transplantation experiments confirm that the target HSPCs can successf?lly home and differentiate,the repair efficiency in vivo are about 2%.Whole-genome high-thro?ghput sequencing analysis showed that no off-target was associated with the targeting system.Conclusion:Our highly efficient universal gene editing repair system of the HBB mutation site has no significant effect on the function of cord blood-HSPCs in vitro and does not induce significant off-target effects,the targets cells have long-term reconstitute ability altho?gh the repair efficiency in vivo decreases after transplantation.Part 3.Repairing the?^CD41-42 HSPCs using the established repair system and verifying gene expressionObjective:Repairing?^CD41-42 HSPCs using the previously established gene targeting repair system,and then analyze the erythroid differentiation ability and HBB gene expression of the repaired cells.Methods:Ficoll density gradient centrif?gation experiment was used to separate fetal liver CD34⁺HSPCs,flow cytometry was used to detect the purity of CD34⁺HSPCs,Sanger sequencing was used to verify changes before and after repair of mutation sites,erythroid differentiation experiments were performed to analyze erythroid differentiation efficiency of?^CD41-42 HSPCs before and after gene targeting,RT-q PCR method was used to detect the HBB m RNA expression of?^CD41-42 HSPCs before and after repair.Res?lts:The isolated fetal liver CD34⁺HSPCs has a purity of 96.7%,the gene targeting repair system can successf?lly repair the?^CD41-42 mutation site,the HSPCs after repair have higher erythroid differentiation efficiency,the HBB m RNA expression of the HSPCs after repair is 6 times higher than that before repair.Conclusion:Our gene-editing repair system can successf?lly repair the?^CD41-42site.The repaired HSPCs have better erythroid differentiation ability and more HBB gene expression.It illustrates that our repairing system has the potential of clinical application.