Dual-AAV delivering split prime editor system for in vivo genome editing

Zhi, Shengyao; Chen, Yuxi; Wu, Guanglan; Wen, Jinkun; Wu, Jinni; Liu, Qianyi; Yang, Li; Kang, Rui; Hu, Sihui; Wang, Jiahui; Liang, Puping; Huang, Junjiu

doi:10.1016/j.ymthe.2021.07.011

Cited by 108 publications

(74 citation statements)

References 48 publications

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“…Notably, however, using the same AAV platform, gene correction levels ranging from 4.1% to 7.4% were measured in RPE65 -associated Leber congenital amaurosis mice after sub-retinal injections ( 58 ). In another recent study, a dual AAV protein trans-splicing system installed a G > T transversion within Dnmt1 at a frequency of 1.71 ± 1.35% 6 weeks after sub-retinal injections in mice ( 59 ). Notwithstanding these important proof-of-concepts, dual AAV designs are complex and relatively inefficient as they operate through only partially controllable intermolecular recombination events for assembling the proper full-length product from the total pool of truncated proteins expressed in co-transduced cells.…”

Section: Resultsmentioning

confidence: 99%

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery

Wang

Liu

Janssen

et al. 2021

Nucleic Acids Research

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Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells with up to 90% efficiencies. Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts.

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Section: Resultsmentioning

confidence: 99%

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery

Wang

Liu

Janssen

et al. 2021

Nucleic Acids Research

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“…However, the large size of prime editors makes them more difficult to deliver in vivo than Cas9 or base editors -a limitation impeding the developing of PEbased gene therapies. Recently, in vivo delivery of prime editors using dual-AAVs has been reported 16,21 .…”

Section: Discussionmentioning

confidence: 99%

“…In a previous study, PE2 had been split at 1005 and 1024 with Rhodothermus marinus (Rma) intein21 , and these two split-PE2 showed ~72.0% and ~75.8% editing efficiency compare with full-length PE2 at HEK3 loci, ~65.6% and 68.4% editing efficiency compare with full-length PE2 at VEGFA loci21 …”

mentioning

confidence: 93%

Development of a flexible split prime editor using truncated reverse transcriptase

Xue

Zheng

Liang

et al. 2021

Preprint

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Prime Editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9, which affects editing efficiency, is limited due to the large size of PE. Furthermore, the potential effect of overexpressing RT in mammalian cells is largely unknown. Here, we developed a compact PE with complete deletion of the RNase H domain of reverse transcriptase (RT), which showed comparable editing to full-length PE. Using compact PE, we tested the effect of 4 different Cas9 split sites and found that the Glu 573 split site supports robust editing (up to 93% of full-length PE). The compact PE, but not PE2, abolished its binding to eRF1 and showed minimal effect on stop codon readthrough, which therefore might reduce the effects on protein biosynthesis. This study identifies a safe and efficient compact PE2 that enables flexible split-PE design to facilitate efficient delivery in vivo and advance the utility of prime editing.

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“…At 6 weeks post-injection, genomic DNA of mouse retina was collected (no cell sorting). This revealed an average editing efficiency of 1.71 ± 1.35% and average indels of 0.17 ± 0.01% in Dnmt1 locus ( Zhi et al, 2021 ). Similarly, a preprint by Jang et al showed an editing efficiency of 1.87% in the Atp7b locus in transduced mouse retina (no cell sorting) and no detectable indels were found.…”

Section: Crispr-based Gene Editing: a Brief Overviewmentioning

confidence: 99%

“…One possibility proposed by the authors is that CBE-mediated indels may occur at a higher rate in retinal cells due to mutual exclusivity between uracil excision pathways and those pathways required for CBE-mediated editing outcomes ( Levy et al, 2020 ). Recently, AAV8-mediated delivery of split-PEs has also been shown to successful edit the Dnmt1 locus in the mouse retina using a CMV promoter ( Zhi et al, 2021 ). In this study, Zhi et al sub-retinally co-injected their split-intein PE along with an AAV8-CMV-GFP reporter at 6 weeks of age and found expression limited to photoreceptors and retinal pigment epithelium (RPE).…”

Section: Crispr-based Gene Editing: a Brief Overviewmentioning

confidence: 99%

Prime Editing for Inherited Retinal Diseases

et al. 2021

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Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation—inherited or de novo—with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.

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Dual-AAV delivering split prime editor system for in vivo genome editing

Cited by 108 publications

References 48 publications

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery

Development of a flexible split prime editor using truncated reverse transcriptase

Prime Editing for Inherited Retinal Diseases

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