Construction Of β-thalassemia Β^CD17-iPSCs And CRISPR-Cas9-based In Vitro Gene Therapy

Objectives:Construction ofβ^CD17-i PSCs forβ-thalassemia;identification of pluripotency,genomic stability and in vitro haematopoietic differentiation capacity of stably transmittedβ^CD17-i PSCs;establishment of an in vitro gene editing system forβ^CD17（A>T）repair based on CRISPR-Cas9 system.Methods:In this research,the Sendai virus vector was used to transduce key transcription factors specifically expressed in ESCs to PBMCs fromβ^CD17pure mutantβ-thalassemia major patients,reprogrammed to obtain non-integratedβ^CD17-i PSCs,cultured without feeder layer to proliferateβ^CD17-i PSCs in vitro,RT-PCR to identify the residual viral vector;AP staining,TRA-60 staining and dd PCR dye to identifyβ^CD17-i PSCs pluripotency,G-chromosome karyotype analysis to identify the genomic stability ofβ^CD17-i PSCs;OP9 cells were co-cultured withβ^CD17-i PSCs in vitro to induce differentiation into erythroid hematopoietic stem progenitors,and flow cytometry detection of recognition antigens on the surface of cultured cells confirmed CD34⁺erythroid hematopoietic stem progenitors.4 pairs of sg RNA electrotransformed 293T cells were created 52bp downstream of the first exon frame of HBB for screening,and PX459 and peg RNA vectors were added to construct the target plasmids,and 293T cells were transfected by liposomes to determine HDR efficiency.Results:After in vitro reprogramming,β^CD17-i PSCs were obtained around 28days later,and subsequent passaged cultures were observed to maintain typical colony state,andβ^CD17-i PSCs without viral vector residues were obtained at P18;β^CD17-i PSCs maintained stemness in passaged cultures and had a normal diploid karyotype（46,XY）;some of theβ^CD17-i PSCs differentiated into CD34⁺cells after co-culture in vitro with OP9 cells;293T cells were screened for efficiently cleaved sg RNA sequences by electrotransfection with the RNP system,inserted into PX459 vector to construct the target plasmid and transfected into 293T cells,and the HDR efficiency was detected to be about 3.68%,in addition,the HDR efficiency was 7.50%when the target plasmid was designed and constructed based on the sg RNA sequence and transfected into 293T cells using the PE2 system.Conclusions:This research successfully constructed non-integratedβ^CD17-i PSCs through in vitro reprogramming,and confirmed the pluripotency,genomic stability and in vitro hematopoietic differentiation ability ofβ^CD17-i PSCs after identification.In addition,an efficient gene editing system for repairingβ^CD17（A>T）was built and the editing efficiency was initially verified,providing an experimental basis for future preclinical investigations using theβ^CD17-i PSCs platform to investigate gene therapy.