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A study published in Science Advances demonstrates the effectiveness of two CRISPR-based gene editing technologies for correcting Duchenne Muscular Dystrophy (DMD) mutations in mice.
Prof Robin Lovell-Badge, Group Leader, The Francis Crick Institute, said:
“This is a complex story, due to the complex nature of the gene encoding Dystrophin and the nature of the mutations within this DMD gene that lead to Duchenne muscular dystrophy, a devastating disease where muscles degenerate and affected individuals (usually boys as it is an X chromosome-linked gene) rarely survive into their 20s and are often incapacitated well before this. But it is also a story that deserves attention. The authors make inventive use of relatively new methods of genome editing, termed Base editing and Prime editing, in order to promote either exon* skipping or reframing, both of which can restore production of a functional Dystrophin protein when the underlying mutation would otherwise lead to a truncated and therefore inactive protein. (After exon skipping, the Dystrophin protein made is a little smaller, but it still functions if the affected part of the protein is within a region containing multiple redundant repeats, which is often the case in Duchenne muscular dystrophy patients.) Because the goal is to treat patients, it was necessary to make use of a viral vector system to introduce the genome editing components. However, the necessary proteins are too large to fit into single AAV (‘adeno-associated viral’) vectors, which are commonly used in gene therapy. The authors made clever use of a newly developed system that allows proteins made by separate vectors to be joined together within the cell, and this was demonstrated to give functional Base and Prime editing proteins. The authors tested the methods in mice and in human muscle cells and could demonstrate that both were able to restore Dystrophin levels sufficiently to rescue muscle fibres to a degree that would be promising as a therapy for patients. However, the authors clearly state that the methods are not there yet, especially the method of delivery of the genome editing components. The amount of AAV vectors they had to use was at least a hundred times higher than that which would be safe. The authors are therefore suitably restrained in their conclusions. A lot more work need to be done to develop the technology before it can be applied to patients – but this paper does represent a very promising step.
“The Olson group had previously reported using earlier, in some ways more straightforward ways of promoting exon skipping using genome editing. This relied on making double strand cuts in DNA which are then repaired by cellular mechanisms but often with small deletions or insertions of DNA. These are OK if the goal is to skip exons, but not if more precise alterations in genes are the aim of the therapy. These earlier methods are also prone to give larger deletions and chromosome rearrangements that could be damaging. However, both Base and Prime editing methods only nick a single strand of the DNA, and these other damaging effects do not occur. This study is therefore a pointer of which way to proceed in other situations where genome editing is being used to correct a defective gene.”
* Many genes, but especially one as large as DMD, is composed of ‘exons’ that carry the code to make the protein, interspersed with non-coding ‘introns’, which have to be removed by ‘splicing’. This splicing doesn’t occur in the DNA, but in the RNA transcript, which is essentially an RNA version of the DNA sequence, and then once spliced, the transcript is referred to as a mature messenger RNA (mRNA), which goes from the nucleus to protein production factories termed ribosomes.
Prof Francesco Muntoni, Director, Dubowitz Neuromuscular Centre & Co-Director MRC Centre for Neuromuscular Diseases at UCL, said:
“This work demonstrate the efficiency of two different techniques (base editors and prime editing) used to correct a deletion mutation of the DMD gene (exon 51), both in vivo and in vitro. This deletion is out-of-frame, and genome editing could be used to induce, for example, a force skipping of exon 50, as the resulting deletion 50-51 is in frame and expected to result in the production of a highly functional protein. The technique of using genome editing to force exon skipping has been used in a variety of dystrophic animal models before by Olson and his group, and several other investigators, with enticing results in vitro and in vivo.
“The novelty of the present work, however, relies on the use of prime editors addressed to splice donors and acceptor regions of DMD gene. This strategy in fact was not applied to DMD before. Prime editors offer the chance to widen the rage of targetable mutations including transversion and translocation and, for the number or separate component that are required to be within the cell at the same time, also lowers the probability of having off target effects. This is essentially a finer “scalpel” that instead of removing an entire exon to induce the re-framing, removed a few nucleotides that are the obstacle for the reframing.
“In this study, 8 predicted off-target sites were analysed upon base editing in this work, and none of them showed sign of off-target editing events. However, an in depth off-target analysis genome wide was not performed.
“The authors recognized the advantages and also limitations of this technology that, being still novel, needs to be further optimised before finding applicability as a potential therapy. A significant limitation for current applicability is the extremely high AAV viral load necessary to deliver the relatively bulky split-intein trans-splicing system, as these high viral loads would preclude systemic clinical translation, in view of the increase concerns for severe adverse events for very high dose of AAV administration. However this is a rapidly evolving field and non-viral delivery is also being successfully developed by others, and could potentially allow the re-administration of the enzymes following muscle growth, which currently cannot be performed for AAV vectors.”
Prof Dominic Wells, Professor in Translational Medicine, Royal Veterinary College and Chair of the Animal Science Group of the Royal Society of Biology, said:
“This is an important proof of principle of the potential use of two refined CRISPR-Cas9 genome editing strategies for the treatment of the X-linked fatal muscle wasting disease called Duchenne muscular dystrophy (DMD). The study was conducted in human cells in culture and in one case in a dystrophic mouse model. The two refined systems are base editing and prime editing, both of which are more strictly targeted genome editors compared to their previous work using CRISPR-Cas9. Consequently, this offers a safer therapeutic approach to the treatment. As is the case in most research, this work builds on the previous work of this group and others but that does not detract from the importance of this proof of principle study.
“The authors are very clear that substantially more work will be required in order to translate this early work into clinical trials. Indeed, the current study involved no randomisation of mice to different experimental groups, used small sample sizes and the analysis was not done blind to sample identity. They used intramuscular injection volumes that could not be scaled up to larger animal models of DMD, let alone man. Thus, DMD patients need to be aware that this work is at a very early experimental stage but does offer another potential therapeutic strategy that is worth further development.”
Dr Tony Lockett, member of the Rare Disease and Gene Therapy Expert Group of the Faculty of Pharmaceutical Medicine, said:
“This is an encouraging development in mice but is a while away from being in the clinic.
“I have seen previous use of this type gene editing to reduce the expression of over-produced proteins (for example in some liver disease) and there is a CRISPR technology in clinical development for transthyretin amyloidosis (ATTR) and no doubt others. But this is the first paper I have seen that looks at the repair of an under-expressed molecule using this type of editing Adenine-Base Editing (ADE). As such it offers a great deal of promise for other rare genetic disorders – not just DMD, but as a treatment for DMD it has a clear advantage where the missing protein is well understood.
“The work presented is very thorough, and well constructed. It has covered every angle I can think of at this stage of development.
“What is good to my mind is the potential lack of off target effects. This is important safety and ethically. The use of CRISPR in somatic cells in this way does not raise ethical issues to my mind, but there are some potential issues. One of the technologies used in genetic disorders and CRISPR cannot be used in women of childbearing potential due to germ cell modification. The use of this technology in DMD (males only) does not raise so much of concern.
“The technology has a long way to go however, the size of the payload to be delivered puts it outside of the systems that I am aware of (the adenovirus systems have a limited packing size). I know some technologies are using liposomes but will require reengineering of the payload. This could create problems as we transit to animals.
“So I think it is an exciting scientific start to managing protein under-expression (e.g. DMD) using a feasible technology (ADE) I am encouraged by it, but it could be a while away from the clinic.”
Dr Yung-Yao Lin, Lecturer, Centre for Genomics and Child Health, Queen Mary University of London, said:
“Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting condition caused by mutations in the DMD gene that encodes a protein called dystrophin. In this proof-of-concept study, scientists elegantly demonstrated the feasibility of two CRISPR-based genome editing strategies with single-nucleotide precision to correct a common DMD mutation. One of the strategies was delivered using an adeno-associated virus to restore expression of dystrophin in a mouse model of DMD.
“This study highlights both opportunities and challenges for developing in vivo genome editing therapeutics that may ultimately be able to treat DMD patients with eligible mutations. In particular, the high viral dose causes safety concerns, which may be overcome using non-viral delivery methods, such as engineered nanoparticles. In addition, evaluation of muscle strength and long-term study will be required to demonstrate the efficacy of the treatment and assess possible immune responses to CRISPR-based editors.”
‘Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing’ by F. Chemello et al. was published in Science Advances at 19:00 UK time on Friday 30th April 2021.
Declared interests
Prof Dominic Wells: “I work at the Royal Veterinary College where the previous work with the CRISPR-Cas9 myoediting was conducted in our beagle cross dystrophic dogs (Amoasii et al., Science 362, 86–91 (2018) – reference 9 in the current paper).. This used the CRISPR-Cas9 induced non-homologous end joining (NHEJ) of double-stranded DNA breaks (DSBs). I was not directly involved in that study.”
Dr Tony Lockett: “None”
None others received