Abstract:Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loaded by vectors could induce high rates of specific site genome editing and correct disease-causing mutations. However, most monogenic genetic diseases such as hemophilia are caused by different mutations dispersed in one gene, instead of an accordant mutation. Vectors developed for correcting specific mutations may not be suited to different mutations at other positions. Site-specific gene addition provides an ideal solution for long-te… Show more
“…Recent studies have successfully used site-specific targeted integration strategies to restore hemostasis in HB mouse models 9 . Site-specific integration of hF9 cDNA has been achieved through nuclease dependent or independent strategies, such as rAAV donor mediated HDR, ZFN or CRISPR/Cas9 stimulated HDR-mediated insertion to ameliorate hemophilia B in mouse models 9, 13, 32 . However, these studies showed a lower HDR efficiency in the adult liver compared with that in newborns.…”
Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.
“…Recent studies have successfully used site-specific targeted integration strategies to restore hemostasis in HB mouse models 9 . Site-specific integration of hF9 cDNA has been achieved through nuclease dependent or independent strategies, such as rAAV donor mediated HDR, ZFN or CRISPR/Cas9 stimulated HDR-mediated insertion to ameliorate hemophilia B in mouse models 9, 13, 32 . However, these studies showed a lower HDR efficiency in the adult liver compared with that in newborns.…”
Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.
“…Recent studies have successfully used site-speci c targeted integration strategies to restore hemostasis in HB mouse models 9 . Site-speci c integration of hF9 cDNA has been achieved through nuclease dependent or independent strategies, such as rAAV donor mediated HDR, ZFN or CRISPR/Cas9 stimulated HDR-mediated insertion to ameliorate hemophilia B in mouse models 9,13,32 . However, these studies showed a lower HDR e ciency in the adult liver compared with that in newborns.…”
Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.
Within less than a decade since its inception, CRISPR-Cas9-based genome editing has been rapidly advanced to human clinical trials in multiple disease areas. Although it is highly anticipated that this revolutionary technology will bring novel therapeutic modalities to many diseases by precisely manipulating cellular DNA sequences, the low efficiency of
in vivo
delivery must be enhanced before its therapeutic potential can be fully realized. Here we discuss the most recent progress of
in vivo
delivery of CRISPR-Cas9 systems, highlight innovative viral and non-viral delivery technologies, emphasize outstanding delivery challenges, and provide the most updated perspectives.
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