Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy

Nelson, Christopher E.; Wu, Yaoying; Gemberling, Matthew; Oliver, Matthew L.; Waller, Matthew A.; Bohning, Joel D.; Robinson-Hamm, Jacqueline N.; Bulaklak, Karen; Rivera, Ruth M. Castellanos; Collier, Joel H.; Asokan, Aravind; Gersbach, Charles A.

doi:10.1038/s41591-019-0344-3

Cited by 322 publications

(325 citation statements)

References 49 publications

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“…Recently, increasing awareness of the fidelity and efficiency of NHEJ repair has led to the development of methods to produce genomic deletions and exogenous sequence insertions using this pathway. As NHEJ is highly active in all phases of the cell cycle, it has been used for gene editing in dividing and non-dividing cells, such as muscles and neurons (12)(13)(14). Targeted deletions are produced by forming two DSBs with loss of the intervening sequence during repair.…”

Section: Introductionmentioning

confidence: 99%

“…Targeted deletions are produced by forming two DSBs with loss of the intervening sequence during repair. The ubiquitous nature of the NHEJ pathway allows for deletions in zygotes, as well as in adult tissue such as in vivo exon deletion in a mouse muscular dystrophy model (14,15). Additionally, exogenously introduced dsDNA donor sequences can efficiently ligate into a single DSB by NHEJ (herein referred to as Insert targeting) (12,13,(16)(17)(18)(19)(20)(21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

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A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

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Lebedin

Rosa

et al. 2020

Preprint

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Precision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and nondividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method 'Replace' editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

Danner

Lebedin

Rosa

et al. 2020

Preprint

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“…While longer term studies are needed in larger animal models, such as canines, a long-term study was recently completed in a mouse model of DMD. The present study utilized systemic AAV delivery of Cas9 in neonatal mice and found a single dose administration resulted in sustained editing and dystrophin protein expression 1 year after treatment [143].…”

Section: Looking Forward: Upcoming Areas For Gene Editor Clinical Trialsmentioning

confidence: 92%

Gene editing and CRISPR in the clinic: current and future perspectives

et al. 2020

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Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

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“…CRISPR cleaves on genomes directly and has shown therapeutic potential in a variety of disease models, in most cases genetic diseases 16-21 . Its therapeutic potential on infectious diseases is promising, but still in its infancy due to lack of strong safety and efficacy evidence supporting for clinical application 22 .…”

Section: Figurementioning

confidence: 99%

Intracorneal delivery of HSV-targeting CRISPR/Cas9 mRNA prevents herpetic stromal keratitis

Yin

Ling

Wang

et al. 2020

Preprint

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1Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current 2 treatments for HSV-1 do not eliminate the virus and are incapable of modulating the virus 3 reservoir. Here, we target HSV-1 genome directly using mRNA-carrying lentiviral particle 4 (mLP) that simultaneously delivers spCas9 mRNA and two viral genes-targeting gRNAs 5 (designated HSV-1-erasing lentiviral particles, HELP). We showed HELP efficiently blocked 6 HSV-1 replication in both acute and recurrent infection models, and prevented occurrence of 7 herpetic stromal keratitis (HSK). We further showed retrograde transportation of HELP from 8 corneas to trigeminal ganglia (TG) where HSV-1 established latency and found evidence of 9 HELP modulating herpes reservoir. Additionally, the potent antiviral activity of HELP was 10 also replicable in human-derived corneas. These results strongly support clinical development 11 of HELP as a new antiviral therapy and may accelerate mRNA-based CRISPR therapeutics. 12

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Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy

Cited by 322 publications

References 49 publications

A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

Gene editing and CRISPR in the clinic: current and future perspectives

Intracorneal delivery of HSV-targeting CRISPR/Cas9 mRNA prevents herpetic stromal keratitis

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