Making gene editing a therapeutic reality

This review discusses current bottlenecks in making CRISPR-Cas9-mediated genome editing a therapeutic reality and it outlines recent strategies that aim to overcome these hurdles as well as the scope of current clinical trials that pioneer the medical translation of CRISPR-Cas9. Additionally, this review outlines the specifics of disease-modifying gene editing in recessive versus dominant genetic diseases with the focus on genetic myopathies that are exemplified by Duchenne muscular dystrophy and myotonic dystrophies.

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In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

Genome Medicine ◽

10.1186/s13073-021-00876-0 ◽

2021 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Menglong Chen ◽

Hui Shi ◽

Shixue Gou ◽

Xiaomin Wang ◽

Lei Li ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Genome Editing ◽

Large Scale ◽

Gene Editing ◽

High Efficiency ◽

Muscle Fibers ◽

Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

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Duchenne muscular dystrophy: genome editing gives new hope for treatment

Postgraduate Medical Journal ◽

10.1136/postgradmedj-2017-135377 ◽

2018 ◽

Vol 94 (1111) ◽

pp. 296-304 ◽

Cited By ~ 2

Author(s):

Vassili Crispi ◽

Antonios Matsakas

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Genome Editing ◽

Genetic Diseases ◽

Wasting Disease ◽

Cardiac Muscles ◽

Dmd Gene ◽

Current Lines ◽

Dystrophin Protein ◽

Potential Therapeutics

Duchenne muscular dystrophy (DMD) is a progressive wasting disease of skeletal and cardiac muscles, representing one of the most common recessive fatal inherited genetic diseases with 1:3500–1:5000 in yearly incidence. It is caused by mutations in the DMD gene that encodes the membrane-associated dystrophin protein. Over the years, many have been the approaches to management of DMD, but despite all efforts, no effective treatment has yet been discovered. Hope for the development of potential therapeutics has followed the recent advances in genome editing and gene therapy. This review gives an overview to DMD and summarises current lines of evidence with regard to treatment and disease management alongside the appropriate considerations.

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Disease-modifying effects of edasalonexent, an NF-κB inhibitor, in young boys with Duchenne muscular dystrophy: Results of the MoveDMD phase 2 and open label extension trial

Neuromuscular Disorders ◽

10.1016/j.nmd.2021.02.001 ◽

2021 ◽

Author(s):

Richard S. Finkel ◽

Erika Finanger ◽

Krista Vandenborne ◽

H. Lee Sweeney ◽

Gihan Tennekoon ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Phase 2 ◽

Open Label ◽

Open Label Extension ◽

Disease Modifying ◽

Extension Trial

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Genotype characterization and delayed loss of ambulation by glucocorticoids in a large cohort of patients with Duchenne muscular dystrophy

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-021-01837-x ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Shu Zhang ◽

◽

Dongdong Qin ◽

Liwen Wu ◽

Man Li ◽

...

Keyword(s):

Clinical Trials ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Hazard Ratio ◽

Large Cohort ◽

Muscle Disease ◽

Clinical Progression ◽

Large Deletions ◽

The Mean ◽

Lower Hazard

Abstract Background Duchenne muscular dystrophy (DMD) is the most common genetic muscle disease in human. We aimed to describe the genotype distribution in a large cohort of Chinese DMD patients and their delayed loss of ambulation by glucocorticoid (GC) treatments. This is to facilitate protocol designs and outcome measures for the emerging DMD clinical trials. Results A total of 1163 patients with DMD were recruited and genotyped. Genotype variations were categorized as large deletions, large duplications, and small mutations. Large deletions were further analyzed for those amenable to exon-skipping therapies. Participants aged 5 years or older were grouped into GC-treated and GC-naïve groups. Clinical progression among different genotypes and their responses to GC treatments were measured by age at loss of ambulation (LOA). Among the mutation genotypes, large deletions, large duplications, and small mutations accounted for 68.79%, 7.14%, and 24.07%, respectively. The mean age at diagnosis was 4.59 years; the median ages at LOA for the GC-naïve, prednisone/prednisolone-treated, and deflazacort-treated groups were 10.23, 12.02, and 13.95 years, respectively. The “deletion amenable to skipping exon 44” subgroup and the nonsense-mutation subgroup had older ages at LOA than the “other deletions” subgroup. Subgroups were further analyzed by both genotypes and GC status. All genotypes showed significant beneficial responses to GC treatment. Deletions amenable to skipping exon 44 showed a lower hazard ratio (0.155). The mean age at death was 18.57 years in this DMD group. Conclusion Genotype variation influences clinical progression in certain DMD groups. Beneficial responses to GC treatment were observed among all DMD genotypes. Compared with other genotypes, deletions amenable to skipping exon 44 had a lower hazard ratio, which may indicate a stronger protective effect of GC treatments on this subgroup. These data are valuable for designing future clinical trials, as clinical outcomes may be influenced by the genotypes.

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Correction of Three Prominent Mutations in Mouse and Human Models of Duchenne Muscular Dystrophy by Single-Cut Genome Editing

Molecular Therapy ◽

10.1016/j.ymthe.2020.05.024 ◽

2020 ◽

Vol 28 (9) ◽

pp. 2044-2055 ◽

Cited By ~ 2

Author(s):

Yi-Li Min ◽

Francesco Chemello ◽

Hui Li ◽

Cristina Rodriguez-Caycedo ◽

Efrain Sanchez-Ortiz ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Genome Editing ◽

Human Models

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Gene Editing With Crispr/Cas9: A New Therapeutic Hope for Duchenne Muscular Dystrophy

MOJ Cell Science & Report ◽

10.15406/mojcsr.2016.03.00050 ◽

2016 ◽

Vol 3 (2) ◽

Author(s):

Sasha Bogdanovich

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Gene Editing

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Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

Blood ◽

10.1182/blood-2021-152429 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3993-3993

Author(s):

Linda Yingqi Lin ◽

Samuele Cancellieri ◽

Jing Zeng ◽

Francesco Masillo ◽

My Anh Nguyen ◽

...

Keyword(s):

Genetic Diversity ◽

Clinical Trials ◽

Genome Editing ◽

Gene Editing ◽

Conflicts Of Interest ◽

African Ancestry ◽

Great Promise ◽

Personal Genome ◽

Hematopoietic Stem ◽

The Impact

Abstract CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

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