Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy shows its not safe due to AAV - and more worryingly Cas9 - integration

2019 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genome Editing ◽  
Mdx Mouse ◽  
Sequencing Data ◽  
Important Distinction ◽  
Adult Mice ◽  
Gene Therapy Application ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD), a monogenic disorder characterized by progressive muscle degeneration, is one of the first diseases being targeted for therapeutic genome editing using nuclease- based methods (CRISPR/ZFN/TALEN). However, safety and persistence remains a concern. Long-term (1 year) persistence and safety of a single intravenous administration of an adeno-associated virus (AAV) and CRISPR was reported in mdx mouse model recently [1]. They reported that ‘AAV-CRISPR is immunogenic when administered to adult mice’, which can be ‘avoided by treating neonatal mice’, and also warned about ‘unintended genome and transcript alterations’. Here, the integration of the Cas9 protein in the exact two locations in the DMD gene which has been edited has been shown based on the same sequencing data (Accid:PRJNA485509). Transcriptomic data also shows Cas9 being expressed. There is an important distinction between AAV and Cas9 integration - while AAV integration can be tolerated, Cas9 integration is a huge, and unacceptable, danger. While there are use cases where the nuclease can be sent as as protein, any gene-therapy application for DMD would require delivery using AAV and the nuclease in a plasmid. So, there is no possible alleviation for this in the future, unless we are willing to accept transgenic humans as a trade-off for curing DMD.

Duchenne muscular dystrophy: genome editing gives new hope for treatment

2018 ◽  
Vol 94 (1111) ◽  
pp. 296-304 ◽  
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.

238 TAILORED PIG MODEL OF DUCHENNE MUSCULAR DYSTROPHY

2012 ◽  
Vol 24 (1) ◽  
pp. 231 ◽  
Author(s):  
N. Klymiuk ◽  
C. Thirion ◽  
K. Burkhardt ◽  
A. Wuensch ◽  
S. Krause ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Nuclear Transfer ◽  
Muscle Disease ◽  
Pig Model ◽  
Mdx Mouse ◽  
Cell Clones ◽  
Fatty Replacement ◽  
Dmd Gene ◽  
Dystrophin Protein

Duchenne muscular dystrophy (DMD) is one of the most common genetic diseases in humans, affecting 1 in 3500 boys. It is characterised by progressive muscle weakness and wasting due to mutations in the dystrophin (DMD) gene resulting in absence of dystrophin protein in skeletal muscle. Although curative treatments are currently not available, genetic and pharmacological approaches are under investigation including early-phase clinical trials. Existing animal models in different species (e.g. mdx mouse, GRMD dog) have been instrumental to understand the pathophysiology of DMD, but have several limitations. Importantly, the causative point mutations (mdx mouse: nonsense mutation; GRMD dog: splice mutation) are different from the most common human mutations (out-of-frame deletion of one or several exons of the DMD gene). We used gene targeting in somatic cells and nuclear transfer to generate a genetically tailored pig model of DMD. A bacterial artificial chromosome (BAC) from the porcine DMD gene was modified by recombineering to replace exon 52, resulting in a frame shift in the transcript. Modified BAC were transfected into male neonatal kidney cells, which were screened by quantitative polymerase chain reaction for replacement of exon 52 in the X-linked DMD gene. Eight of 436 cell clones were successfully targeted and 2 of them were used for nuclear transfer. For each of the cell clones, a pregnancy was established by transfer of cloned embryos into recipient gilts. Four piglets of the first litter were live born and killed within 48 h and tissue samples were processed for histological characterisation. Two piglets of the second litter died during birth due to obstetric complications, whereas the other 2 piglets were delivered by Caesarean section and raised in an artificial feeding system. Their serum creatine kinase (CK) levels were grossly elevated. Although both piglets showed reduced mobility compared with age-matched controls, they were able to move and feed on their own. Immunofluorescence staining of dystrophin was negative in muscle fibres of DMD mutant piglets and the complete absence of dystrophin protein was confirmed by immunoblot analysis. Histological examination of biceps femoris muscle from DMD mutant pigs showed a degenerative myopathy with fibre size variation, rounded fibres, central nuclei, fibrosis and fatty replacement of muscle tissue mimicking the hallmarks of the human disease. In conclusion, we generated the first pig model for a genetic muscle disease. The DMD mutant pig appears to be a bona fide model of the human dystrophy as ascertained by absence of the dystrophin protein, elevated serum CK levels and early degenerative changes on muscle histology. Because deletion of exon 52 is one of the most frequent mutations found in human DMD, the exon 52 mutated DMD pig represents an excellent model for testing targeted genetic treatments. This study was supported by the Bayerische Forschungsstiftung.

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

2019 ◽  
Vol 25 (3) ◽  
pp. 427-432 ◽  
Author(s):  
Christopher E. Nelson ◽  
Yaoying Wu ◽  
Matthew P. Gemberling ◽  
Matthew L. Oliver ◽  
Matthew A. Waller ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genome Editing ◽  
Term Evaluation

scholarly journals Comparison of Long-term Ambulatory Function in Patients with Duchenne Muscular Dystrophy Treated with Eteplirsen and Matched Natural History Controls

2021 ◽  
pp. 1-11
Author(s):  
Jerry R. Mendell ◽  
Navid Khan ◽  
Nanshi Sha ◽  
Helen Eliopoulos ◽  
Craig M. McDonald ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Gene Mutations ◽  
Exon Skipping ◽  
Term Treatment ◽  
Long Term Treatment ◽  
The Difference ◽  
Dmd Gene ◽  
6 Minute Walk Test

Background: Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by DMD gene mutations. A relationship between exon skipping and dystrophin production in exon 51-amenable patients treated with eteplirsen (EXONDYS 51 ®) is established. Once-weekly eteplirsen significantly increased dystrophin, with slower decline in ambulatory function compared to baseline. Long-term treatment with eteplirsen leads to accumulation of dystrophin over time and observed functional benefits in patients with DMD. Objective: Compare long-term ambulatory function in eteplirsen-treated patients versus controls. Methods: Study 201/202 included 12 eteplirsen-treated patients assessed twice/year for ambulatory function over 4 years. Ambulatory evaluations (6-minute walk test [6MWT], loss of ambulation, and North Star Ambulatory Assessment [NSAA]) were compared with matched controls from Italian Telethon and Leuven registries. Results: At Years 3 and 4, eteplirsen-treated patients demonstrated markedly greater mean 6MWT than controls (difference in change from baseline of 132 m [95%CI (29, 235), p = 0.015] at Year 3 and 159 m [95%CI (66, 253), p = 0.002] at Year 4). At Year 4, a significantly greater proportion of eteplirsen-treated patients were still ambulant versus controls (10/12 vs 3/11; p = 0.020). At Year 3, eteplirsen-treated patients had milder NSAA decline than controls (difference in change from baseline of 2.6, 95%CI [-6, 11]), however, the difference was not statistically significant; Year 4 control NSAA data were not available. Conclusion: In this retrospective matched control study, eteplirsen treatment resulted in attenuation of ambulatory decline over a 4-year observation period, supporting long-term benefit in patients with DMD.

scholarly journals Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy

2021 ◽  
Author(s):  
Ayhan Atmanli ◽  
Andreas C Chai ◽  
Miao Cui ◽  
Zhaoning Wang ◽  
Takahiko Nishiyama ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Dilated Cardiomyopathy ◽  
Genome Editing ◽  
Rna Sequencing ◽  
Reading Frame ◽  
Transcriptional Dysregulation ◽  
Cardiac Abnormalities ◽  
Dmd Gene ◽  
Novel Therapeutic

Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of DMD patients, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional and transcriptional abnormalities in DMD-associated cardiac disease. Methods and Results: We generated induced pluripotent stem cells (iPSCs) from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD (cDMD) iPSC lines, wherein the DMD open reading frame was restored via reframing (RF) or exon skipping (ES). While DMD cardiomyocytes (CMs) demonstrated morphologic, structural and functional deficits compared to control CMs, CMs from both cDMD lines were similar to control CMs. Bulk RNA-sequencing of DMD CMs showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in cDMD CMs. We then corrected dysfunctional DMD CMs by adenoviral delivery of Cas9/gRNA and showed that correction of DMD CMs post-differentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in CMs and fibroblasts, which in corrected mice was reduced to similar levels as wildtype mice. Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in iPSC-CMs and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.

scholarly journals Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence

2022 ◽  
Vol 12 ◽  
Author(s):  
Arianna Manini ◽  
Elena Abati ◽  
Andi Nuredini ◽  
Stefania Corti ◽  
Giacomo Pietro Comi
Keyword(s):  
Gene Therapy ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genetic Material ◽  
Premature Death ◽  
Gene Replacement ◽  
Neuromuscular Disorder ◽  
Adeno Associated Virus ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is an X-linked recessive, infancy-onset neuromuscular disorder characterized by progressive muscle weakness and atrophy, leading to delay of motor milestones, loss of autonomous ambulation, respiratory failure, cardiomyopathy, and premature death. DMD originates from mutations in the DMD gene that result in a complete absence of dystrophin. Dystrophin is a cytoskeletal protein which belongs to the dystrophin-associated protein complex, involved in cellular signaling and myofiber membrane stabilization. To date, the few available therapeutic options are aimed at lessening disease progression, but persistent loss of muscle tissue and function and premature death are unavoidable. In this scenario, one of the most promising therapeutic strategies for DMD is represented by adeno-associated virus (AAV)-mediated gene therapy. DMD gene therapy relies on the administration of exogenous micro-dystrophin, a miniature version of the dystrophin gene lacking unnecessary domains and encoding a truncated, but functional, dystrophin protein. Limited transgene persistence represents one of the most significant issues that jeopardize the translatability of DMD gene replacement strategies from the bench to the bedside. Here, we critically review preclinical and clinical studies of AAV-mediated gene therapy in DMD, focusing on long-term transgene persistence in transduced tissues, which can deeply affect effectiveness and sustainability of gene replacement in DMD. We also discuss the role played by the overactivation of the immune host system in limiting long-term expression of genetic material. In this perspective, further studies aimed at better elucidating the need for immune suppression in AAV-treated subjects are warranted in order to allow for life-long therapy in DMD patients.

scholarly journals Dental pulp stem cells can improve muscle dysfunction in animal models of Duchenne muscular dystrophy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuko Nitahara-Kasahara ◽  
Mutsuki Kuraoka ◽  
Posadas Herrera Guillermo ◽  
Hiromi Hayashita-Kinoh ◽  
Yasunobu Maruoka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Dental Pulp ◽  
Systemic Treatment ◽  
Dental Pulp Stem Cells ◽  
Mri Findings ◽  
Systemic Injection ◽  
Therapeutic Benefits

Abstract Background Duchenne muscular dystrophy (DMD) is an inherited progressive disorder that causes skeletal and cardiac muscle deterioration with chronic inflammation. Dental pulp stem cells (DPSCs) are attractive candidates for cell-based strategies for DMD because of their immunosuppressive properties. Therefore, we hypothesized that systemic treatment with DPSCs might show therapeutic benefits as an anti-inflammatory therapy. Methods To investigate the potential benefits of DPSC transplantation for DMD, we examined disease progression in a DMD animal model, mdx mice, by comparing them with different systemic treatment conditions. The DPSC-treated model, a canine X-linked muscular dystrophy model in Japan (CXMDJ), which has a severe phenotype similar to that of DMD patients, also underwent comprehensive analysis, including histopathological findings, muscle function, and locomotor activity. Results We demonstrated a therapeutic strategy for long-term functional recovery in DMD using repeated DPSC administration. DPSC-treated mdx mice and CXMDJ showed no serious adverse events. MRI findings and muscle histology suggested that DPSC treatment downregulated severe inflammation in DMD muscles and demonstrated a milder phenotype after DPSC treatment. DPSC-treated models showed increased recovery in grip-hand strength and improved tetanic force and home cage activity. Interestingly, maintenance of long-term running capability and stabilized cardiac function was also observed in 1-year-old DPSC-treated CXMDJ. Conclusions We developed a novel strategy for the safe and effective transplantation of DPSCs for DMD recovery, which included repeated systemic injection to regulate inflammation at a young age. This is the first report on the efficacy of a systemic DPSC treatment, from which we can propose that DPSCs may play an important role in delaying the DMD disease phenotype.

Have Duchenne Muscular Dystrophy Patients an Increased Cancer Risk?

2021 ◽  
pp. 1-5
Author(s):  
Gian Luca Vita ◽  
Luisa Politano ◽  
Angela Berardinelli ◽  
Giuseppe Vita
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Systematic Investigation ◽  
Mdx Mice ◽  
True Incidence ◽  
Patients With Cancer ◽  
Dmd Gene ◽  
Age Range ◽  
Prevalence Of Cancer

Background: Increasing evidence suggests that Duchenne muscular dystrophy (DMD) gene is involved in the occurrence of different types of cancer. Moreover, development of sarcomas was reported in mdx mice, the murine model of DMD, in older age. So far, nine isolated DMD patients were reported with concomitant cancer, four of whom with rhabdomyosarcoma (RMS), but no systematic investigation was performed about the true incidence of cancer in DMD. Methods: All members of the Italian Association of Myology were asked about the occurrence of cancer in their DMD patients in the last 30 years. Results: Four DMD patients with cancer were reported after checking 2455 medical records. One developed brain tumour at the age of 35 years. Two patients had alveolar RMS at 14 and 17 years of age. The fourth patient had a benign enchondroma when 11-year-old. Conclusion: Prevalence of cancer in general in the Italian DMD patients does not seem to be different from that in the general population with the same age range. Although the small numbers herein presented do not allow definitive conclusion, the frequent occurrence of RMS in DMD patients raises an alert for basic researchers and clinicians. The role of DMD gene in cancer merits further investigations.

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