Prime editing optimized RTT permits the correction of the c.8713C&gt;T mutation in DMD gene

Mbakam, Cédric Happi; Rousseau, Joël; Lu, Yaoyao; Bigot, Anne; Mamchaoui, Kamel; Mouly, Vincent; Tremblay, Jacques P.

doi:10.1016/j.omtn.2022.09.022

Cited by 15 publications

(12 citation statements)

References 39 publications

(56 reference statements)

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“…These weak results might be due to the fact that this approach did not involve mutating the PAM. Mutating the PAM during Prime editing has been shown to increase Prime editing efficiency ( 21 ).…”

Section: Resultsmentioning

confidence: 99%

“…This has been shown to significantly increase the Prime editing efficiency ( 29 ). In addition to mutating the PAM sequence and PE3 approach, one or two additional synonymous mutations were also added on either side of the intended modification to increase the editing efficiency ( 21 , 30 ). Our group has recently shown that the addition of synonymous mutations has the potential to increase the Prime editing efficacy in the DMD gene by 9.6-fold ( 21 ).…”

Section: Resultsmentioning

confidence: 99%

“…In addition to mutating the PAM sequence and PE3 approach, one or two additional synonymous mutations were also added on either side of the intended modification to increase the editing efficiency ( 21 , 30 ). Our group has recently shown that the addition of synonymous mutations has the potential to increase the Prime editing efficacy in the DMD gene by 9.6-fold ( 21 ). These epegRNAs were configured to change the GT of the splice donor site of exon 51 to either CA (SubSpD51−1,2, and 3), CC (SubSpD51−4,5,6,12, and 16), CG (SubSpD51−7,8,9,10,11, and 15), or TG (SubSpD51−13 and 14).…”

Section: Resultsmentioning

confidence: 99%

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Prime editing strategies to mediate exon skipping in DMD gene

Happi Mbakam,

Roustant,

Rousseau

et al. 2023

Front. Med.

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Duchenne muscular dystrophy is a rare and lethal hereditary disease responsible for progressive muscle wasting due to mutations in the DMD gene. We used the CRISPR-Cas9 Prime editing technology to develop different strategies to correct frameshift mutations in DMD gene carrying the deletion of exon 52 or exons 45 to 52. With optimized epegRNAs, we were able to induce the specific substitution of the GT nucleotides of the splice donor site of exon 53 in up to 32% of HEK293T cells and 28% of patient myoblasts. We also achieved up to 44% and 29% deletion of the G nucleotide of the GT splice site of exon 53, as well as inserted 17% and 5.5% GGG between the GT splice donor site of exon 51 in HEK293T cells and human myoblasts, respectively. The modification of the splice donor site for exon 51 and exon 53 provoke their skipping and allowed exon 50 to connect to exon 53 and allowed exon 44 to connect to exon 54, respectively. These corrections restored the expression of dystrophin as demonstrated by western blot. Thus, Prime editing was used to induce specific substitutions, insertions and deletions in the splice donor sites for exons 51 and 53 to correct the frameshift mutations in DMD gene carrying deletions of exon 52 and exons 45 to 52, respectively.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Prime editing strategies to mediate exon skipping in DMD gene

Happi Mbakam,

Roustant,

Rousseau

et al. 2023

Front. Med.

Self Cite

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show abstract

“…Frontiers in Genome Editing frontiersin.org Kweon et al, 2021;Nelson et al, 2021;Liu N. et al, 2022;Happi Mbakam et al, 2022;Hong et al, 2022;Kweon et al, 2022;Levesque et al, 2022;Peterka et al, 2022;Schene et al, 2022;Simon et al, 2022;Tao et al, 2022;Velimirovic et al, 2022;Wang et al, 2022;Zhang et al, 2022;Zhuang et al, 2022). We and others have previously utilized plasmid-based prime editors allowing stable integration of the system into the genome of human cells using piggyBac transposase, taking advantage of the prolonged expression to achieve highly effective prime editing (Eggenschwiler et al, 2021;Wolff et al, 2021).…”

Section: Delivery Is the Keymentioning

confidence: 99%

Prime editing in hematopoietic stem cells—From ex vivo to in vivo CRISPR-based treatment of blood disorders

Wolff

Mikkelsen

2023

Front. Genome Ed.

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Prime editing of human hematopoietic stem cells has the potential to become a safe and efficient way of treating diseases of the blood directly in patients. By allowing site-targeted gene intervention without homology-directed repair donor templates and DNA double-stranded breaks, the invention of prime editing fuels the exploration of alternatives to conventional recombination-based ex vivo genome editing of hematopoietic stem cells. Prime editing is as close as we get today to a true genome editing drug that does not require a separate DNA donor. However, to adapt the technology to perform in vivo gene correction, key challenges remain to be solved, such as identifying effective prime editing guide RNAs for clinical targets as well as developing efficient vehicles to deliver prime editors to stem cells in vivo. In this review, we summarize the current progress in delivery of prime editors both in vitro and in vivo and discuss future challenges that need to be adressed to allow in vivo prime editing as a cure for blood disorders.

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“…PegRNA directs the Cas9 nickase to a specific target locus and encodes a specific editing sequence of interest. Multiple studies have reported the use of PEs for in vivo gene editing to treat diseases [23][24][25] (Figure 1). For example, Liu et al injected PE2 to correct the pathogenic mutation in the Alpha-1 antitrypsin deficiency (AATD) mouse model.…”

Section: Genetic Diseasesmentioning

confidence: 99%

Gene therapy for pediatric genetic kidney diseases

Song

Sun

et al. 2023

Pediatric Discovery

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Genetic kidney disease is the main cause of chronic kidney disease in children. While the pathogenic genes associated with most genetic kidney diseases have been identified, the underlying mechanisms of disease initiation remain ambiguous, and effective treatment modalities are limited. Gene therapy has emerged as a promising approach for treating genetic diseases. Several gene therapy drugs have been successfully launched to treat genetic diseases affecting the eyes and muscles. However, the development of gene therapy agents for kidney diseases lags behind that of other genetic disorders. In this review, we first comprehensively summarize the main gene therapy strategies. Next, we provide an overview of current treatment options as well as the latest research on gene therapy approaches for pediatric genetic kidney diseases with a particular emphasis on Alport syndrome and polycystic kidney disease. Finally, we discuss the challenges and potential solutions for gene therapy of pediatric genetic kidney diseases.

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Prime editing optimized RTT permits the correction of the c.8713C>T mutation in DMD gene

Cited by 15 publications

References 39 publications

Prime editing strategies to mediate exon skipping in DMD gene

Prime editing strategies to mediate exon skipping in DMD gene

Prime editing in hematopoietic stem cells—From ex vivo to in vivo CRISPR-based treatment of blood disorders

Gene therapy for pediatric genetic kidney diseases

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