Single AAV-mediated CRISPR-Nme2Cas9 efficiently reduces mutant hTTR expression in a transgenic mouse model of transthyretin amyloidosis

Wen, Jinkun; Cao, Tianqi; Wu, Jinni; Chen, Yuxi; Zhi, Shengyao; Huang, Yanming; Zhen, Peilin; Wu, Guanglan; Aagaard, Lars; Zhong, Jun; Liang, Puping; Huang, Junjiu

doi:10.1016/j.ymthe.2021.05.010

Cited by 18 publications

(13 citation statements)

References 51 publications

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“…Nme2Cas9 is known to be highly accurate in nuclease based editing in cells [27,30,39], and our mismatch scanning experiments ( Figure 1e ) strongly suggest that the same will be true in vivo . To evaluate potential Cas9-dependent off-target effects in the AAV9-injected mice, we searched for genome-wide off-target sites for Nme2Cas9 using Cas-OFFinder [74], allowing for up to 6 mismatches.…”

Section: Resultssupporting

confidence: 54%

“…We did not detect any above-background level of indel, indicating that single-AAV delivered Nme2-ABE8e can install precise editing in vivo without generating unwanted indels (Figure 5c). Nme2Cas9 is known to be highly accurate in nuclease based editing in cells [27,30,39], and our mismatch scanning experiments (Figure 1e) strongly suggest that the same will be true in vivo. To evaluate potential Cas9-dependent off-target effects in the AAV9-injected mice, we searched for genome-wide off-target sites for Nme2Cas9 using Cas-OFFinder [74], allowing for up to 6 mismatches.…”

Section: Hydrodynamic Injection Of Single-aav Vector Plasmids Corrects the Disease Mutation And Phenotype In An Adult Mouse Model Of Ht1supporting

confidence: 55%

Section: Hydrodynamic Injection Of Single-aav Vector Plasmids Corrects the Disease Mutation And Phenotype In An Adult Mouse Model Of Ht1supporting

confidence: 55%

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Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Zhang

Bamidele

et al. 2021

Preprint

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Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

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Section: Resultssupporting

confidence: 54%

Section: Hydrodynamic Injection Of Single-aav Vector Plasmids Corrects the Disease Mutation And Phenotype In An Adult Mouse Model Of Ht1supporting

confidence: 55%

Section: Hydrodynamic Injection Of Single-aav Vector Plasmids Corrects the Disease Mutation And Phenotype In An Adult Mouse Model Of Ht1supporting

confidence: 55%

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Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Zhang

Bamidele

et al. 2021

Preprint

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Section: Hydrodynamic Injection Of Single-aav Vector Plasmids Correct...supporting

confidence: 57%

“…It has been shown that the major source of DNA off-target base editing is Cas9-dependent [37,38], caused by Cas9 binding and unwinding at near-cognate sequences. Because Nme2Cas9 is highly accurate during nuclease-driven editing in cells and in vivo [27,30,39], we hypothesized that Nme2-ABE8e would exhibit similar accuracy advantages relative to Spy-ABE8e. As an initial test, we systematically investigated the tolerance for nucleotide mismatches between the guide and the target sequence for the two effectors.…”

Section: Development Of Nme2-abe and Comparison Of Editing Windows An...mentioning

confidence: 99%

Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector

et al. 2022

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Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we show that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can efficiently edit mouse genomic loci and revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

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CRISPR‐based gene therapy for wet age‐related macular degeneration in mouse model

Xie,

Chen,

et al. 2024

Clinical and Translational Dis

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Wet age‐related macular degeneration (AMD) is a common cause of vision loss in the elderly. It is characterised by choroidal neovascularisation (CNV), caused by overexpression of vascular endothelial growth factor (VEGF), resulting in abnormal vessel proliferation. Current clinical management predominantly relies on anti‐VEGF agents, which require frequent and costly injections. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising strategy for permanently suppressing angiogenesis by targeting the VEGF‐related pathway. Increased research suggests that disrupting this pathway holds potential for preventing CNV progression. This review provides an overview of the aetiology, classification and pathophysiology of wet AMD, followed by a concise summary of current gene editing research using the CRISPR/Cas system via viral vector delivery strategies to target ocular pro‐angiogenic factors, including Hif‐1α, VEGF and VEGFR. The importance of timely targeting of VEGFA is emphasised and the challenges associated with gene editing therapies are also highlighted.

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Single AAV-mediated CRISPR-Nme2Cas9 efficiently reduces mutant hTTR expression in a transgenic mouse model of transthyretin amyloidosis

Cited by 18 publications

References 51 publications

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector

CRISPR‐based gene therapy for wet age‐related macular degeneration in mouse model

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