Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

Zhang, Jianping; Cheng, Xinxin; Zhao, Mei; Li, Guohua; Xu, Jing; Zhang, Feng; Yin, Mengdi; Meng, Feiying; Dai, Xinyue; Fu, Ya-Wen; Yang, Zhouxin; Arakaki, Cameron; Su, Ruijun; Wen, Wei; Wang, Wentian; Chen, Wanqiu; Choi, Hannah; Wang, Charles; Gao, Guangping; Zhang, Lei; Cheng, Tao; Zhang, Xiaobing

doi:10.1186/s13059-019-1907-9

Cited by 55 publications

(58 citation statements)

References 53 publications

(70 reference statements)

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“…We used a triple plasmid transfection protocol to produce recombinant AAV vectors as detailed previously ( 42 , 43 ). In brief, HEK293T cells at the 80–90% confluency were transfected with the complex of PEI (polyethylenimine) MAX 40K (Polysciences) and AAV plasmids at a mass ratio of 2:1. pAAV-Helper (Cell Biolabs), pR2C6 (AAV6 capsid vector) (Cell Biolabs), pAAV-HDR (transgene vector construct) were added at a ratio of 2:1:1.…”

Section: Methodsmentioning

confidence: 99%

“…To deplete iodixanol in the final AAV products, we washed the vectors twice with PBS–0.01% Pluronic F68 using the Vivaspin 20 centrifugal concentrators (MWCO 100 kDa). The AAV6 vector titers were determined by qPCR analysis using the vector plasmids as controls ( 42 ). Individual vector-specific primers (also for amplicon sequencing) were used.…”

Section: Methodsmentioning

confidence: 99%

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Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing

Dai

Wang

et al. 2021

Nucleic Acids Research

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Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing

Dai

Wang

et al. 2021

Nucleic Acids Research

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“…In a separate study by Zhang et al, CRISPR-Cas9 introduced using HD was used to correct mouse models of Hemophilia A [ 212 ]. The authors delivered a donor template that would lead to the integration of B domain-deleted FVIII (BDDF8) into the Alb locus along with CRISPR components via hydrodynamic delivery and observed 0.1% and 2% knockin efficiencies with circular and linearized plasmid donors, respectively.…”

Section: Delivery Strategies For Therapeutic Applicationsmentioning

confidence: 99%

Delivery Approaches for Therapeutic Genome Editing and Challenges

Ates

Rathbone

Stuart

et al. 2020

Genes

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Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells.

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“…In addition, gene editing approaches are being developed and first results in a haemophilia mouse model of FVIII gene editing by co‐delivery of three AAV8 vectors carrying Cas9, SgRNA or BDDF8 donor have shown persistent restoration of FVIII activity for over one year 47 …”

Section: Gene Therapy For Haemophilia In Chinamentioning

confidence: 99%

Hemophilia gene therapy—New country initiatives

2020

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Gene therapy is an opportunity for haemophilia patients to receive a one‐time treatment and have lasting factor levels for years or decades instead of dependence on repeated administration within short intervals and on sustained supply of drug. Great strides have been made in the development of gene therapy for haemophilia in the last decade. Adeno‐associated virus (AAV) vector–mediated gene transfer in haemophilia A and B has entered the phase III trial stage. Gene transfer by lentiviral vector or gene editing technologies using factor VIII (FVIII) or IX (FIX) genes are now entering clinical evaluation. It is expected that the first FVIII and FIX gene therapy products will soon be approved and distributed in major markets. Global access to gene therapy is a critical goal. This review presents new and ongoing efforts towards this goal in countries other than North America and Europe. In Japan, researchers, regulators and funders have established a promising gene therapy development platform for multiple diseases including haemophilia. Decades of scientific and clinical research in haemophilia gene therapy in China have led to a recently registered clinical trial of AAV‐mediated gene therapy for haemophilia B. Other countries are in earlier phases of building gene therapy programmes or participate in international trials. A phase 2 feasibility trial of AAV‐mediated FIX gene therapy in low‐ and middle‐income countries aims to demonstrate that gene therapy could become available in resource‐constrained socio‐economic settings. The different strategies for establishing gene therapy provide opportunities for closing the global gap in haemophilia care.

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Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

Cited by 55 publications

References 53 publications

Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing

Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing

Delivery Approaches for Therapeutic Genome Editing and Challenges

Hemophilia gene therapy—New country initiatives

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