| Induced pluripotent stem cells(iPSCs)have demonstrated significant potential in basic research and regenerative medicine due to their infinite expansion and multilineage differentiation capabilities.To fully harness the immense potential of iPSCs,CRISPRCas9 gene editing is often employed.However,many disease-causing genes are located within closed chromatin regions in iPSCs,limiting the editing efficiency of CRISPR-Cas9 technology at these sites.Therefore,we attempted to address the technical bottleneck of gene editing in closed loci of iPSCs.By treating electroporated iPSCs with HDAC inhibitors,we found that the efficiency of non-homologous end joining(NHEJ)and homologous recombination(HDR)at closed loci increased by 1.5-fold and 3-fold,respectively.Furthermore,HDAC inhibitors also enhanced the expression levels of Cas9 nuclease and sgRNA editing components in iPSCs,thereby improving the overall cutting efficiency of the genome.Secondly,off-target cleavage of CRISPR-Cas9 gene editing technology is a critical issue that needs to be addressed in the clinical application.We optimized the widely-used GUIDE-seq method and named the new method as OliTag-seq.In OliTag-seq,the fulllength insertion efficiency of dsODN in Cas9-induced double-strand breaks(DSBs)increased by 1.7-fold,and the use of a three-primer mix in a single PCR round significantly simplified the library construction process.Additionally,we further confirmed that chromatin accessibility affects Cas9’s specific cutting,and iPSCs exhibit superior offtarget recognition capabilities due to their loose chromatin.Moreover,the constitutive expression of Cas9 and transient chromatin opening by HDAC inhibitors further improved off-target recognition.Using OliTag-seq,we identified several new off-target sites in sgRNAs related to CAR-T cell therapy.Furthermore,considering the current immature development of CRISPR-Cas9 technology and the limited number of related gene therapy clinical trials,we focused on the traditional lentiviral vector therapy.We systematically optimized Bluebird Bio’s BB305 lentiviral vector for β-thalassemia,silenced potential transcription termination signals,reduced the LCR enhancer element from 2.6 kb to 1.9 kb,and designed an HBG2 reactivation promoter,resulting in a novel HBB lentiviral vector.Compared to the BB305 vector,the new vector exhibited a 6-fold increase in titer and a more than 40%increase inβ-globin expression levels and exhibited improved therapeutic effects in mice,potentially significantly reducing the cost of gene therapy for β-thalassemia.Lastly,we analyzed the integration safety of the novel HBB lentiviral vector in the genome and preliminarily explored data analysis methods for lentiviral integration sites.Following the methods mentioned in INSPIIRED,we introduced a blocking oligonucleotide during the PCR process and tested multiple PCR conditions to maximally enrich 3’-LTR and nearby genomic sequences at the integration sites.By modifying the OliTag-seq workflow to analyze the HBB lentiviral vector integration site data,we found that the integration sites were abundant in genes such as HBG2,TBC1D5,and MROH1,and were mostly concentrated in intronic regions,consistent with the known integration preference of lentiviral vectors.In summary,we conducted several technical developments in the areas of stem cell gene editing and gene therapy research,addressing to some extent the technical challenges of iPSC non-open locus gene editing.We also systematically optimized the BB305 lentiviral vector,improving both vector titer and β-globin expression levels.Additionally,we developed analysis workflows to address the safety concerns of CRISPR-Cas9 gene editing and lentiviral vector gene therapy.We hope that our research can provide guidance for gene therapy in β-thalassemia and cancer immunotherapy,and we look forward to the clinical or industrial translation of our technical achievements in future. |
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