From theory to therapy

It has become the toast of the genomics community – but can CRISPR really translate to the clinic? There are very promising signs says Dermot Martin…

Duchenne Muscular Dystrophy (DMD) is a devastating genetic disease which affects about 1 out of 3,500 mostly males and is caused by mutations in the dystrophin gene. The stark reality for victims and their relatives is a life expectancy only in the mid-twenties.

Three decades after the discovery of the human gene that causes DMD, little practical progress has been made in finding a new treatments or therapies. It was hoped that the discovery of dystrophin, the rod-shaped protein which connects the cytoskeleton of a muscle fibre to the surrounding cell matrix through the cell membrane would be a crucial breakthrough.

Understanding the role of dystrophin and its structure sparked a burst of research energy invested in understanding the processes involved in DMD

For the architecture of a human muscle cell, dystrophin is as vital as any re-enforced steel joist to the integrity of a building. Researchers were able to link a lack of dystrophin to the collapse of the structure of muscle tissue and the relentless degeneration of the muscle fibres as calcium ions from the bloodstream invade the tissue. DMD victims usually succumb to the disease in their mid-twenties as the heart muscle suffers this fate. Understanding the role of dystrophin and its structure sparked a burst of research energy invested in understanding the processes involved in DMD. We now know the condition arises from a recessive, fatal, X-linked gene occurring at a frequency of about 1 in 3,500 new-born males. Becker MD is a milder allelic form of the condition.

In general, DMD patients carry mutations or deletions, which cause premature translation termination while in BMD patients dystrophin is reduced either in molecular weight (derived from in-frame deletions) or in expression level. The dystrophin gene is highly complex, containing at least eight independent, tissue-specific promoters and two poly A-addition sites. Dystrophin RNA is differentially spliced, producing a range of different transcripts, encoding a large set of protein isoforms.

New battle fronts But now the rapid development of CRISPR/Cas9 editing system (clustered regularly interspaced short palindromic repeats) has opened a new battle front in the war against DMD. It is now beginning to deliver vital new data, at very low cost, about how the gene operates as a well as route into mutant gene therapy. CRISPR is often described in simple terms as genetic cut and paste technique applied to the genetic code. It has been hailed as the biggest breakthrough in gene splicing and editing technology this century and is becoming the ‘go-to’ method for gene manipulation.

Central to CRISPR are two molecules, which effectively work as a pair of scissors and a microscopic sat nav

The technique evolved from studies of antibiotics and the intricate mechanisms bacteria use to build resistance specifically to attack by viruses. Central to CRISPR are two molecules, which effectively work as a pair of scissors and a microscopic sat nav. The protein called Cas9 acts as the ‘scissors’ which can chop up DNA. But precisely where it does so is directed by a short strand of piece of DNA’s molecular cousin, ribonucleic acid (RNA).

These two components evolved in bacteria to protect them against invading viruses. It works by helping bacteria identify and destroy the viruses’ own DNA. Researchers worked out how to isolate and adapt these ‘tools’ and quickly realised they could use them to edit any gene they wished, in any cell they like, with absolute precision. First, the RNA ‘sat nav’ precisely matches up with a particular stretch of DNA. And it brings the Cas9 molecule along with it, allowing the scissors to cut at that exact point in the DNA sequence.

CRISPR’s is a quantum leap in terms of research because it allows us to engineer bespoke versions of the RNA ‘sat nav’, allowing, in theory, the Cas9 to be directed to any gene we might wish. The CRISPR system has been popularly described as a cellular version of the ‘find and replace’ tool on a word processor. Once Cas9 has cut the DNA, the cell’s built-in repair machinery swings into action. Researchers can use this response to disrupt the gene that has been cut, essentially switching it off to see study the effects.

There are dozens of laboratories around the world now working on the disease but three CRISPR centred studies in mice are causing particular attention

They can also do more sophisticated experiments which change the DNA code precisely. They can make ‘spelling mistakes’ in a gene, like certain faults seen in cancer cells, which alter the gene’s function rather than merely scrambling it as they did before CRISPR. This type of work is essential for tackling the DMD conundrum. There are dozens of laboratories around the world now working on the disease but three CRISPR centred studies in mice are causing particular attention. In one case, the CRISPR/Cas9 system targeted the point mutation in exon 23 of the mdx mouse that creates a premature stop codon and serves as a representative model of DMD.

A clinical path These studies are important because they lay the groundwork for clinical translation, addressing many of the critical questions that have been raised regarding this system of gene correction. Each of the three studies have already demonstrated the value of using a what’s called a two-vector system of AAV-CRISPR rather than single vectors for both the guide RNA and the Cas9 nuclease.

For instance, the cDNAs from staphylococcus aureus Cas9 and streptococcus pyogenes Cas9 were both effective in these in vivo pre-clinical studies and delivery of vectors using either adeno-associated virus AAV serotypes, AAV9 or AAV8, performed well. Although genomic editing within the germline is not currently feasible in humans, clinical studies using these AAV serotypes have proved to be safe, demonstrating at least feasibility for human application. However, genomic editing could, in principle, be envisioned within postnatal cells in vivo if certain technical challenges could be overcome.

For instance, there is a need for appropriate somatic cell delivery systems capable of directing the components of the CRISPR/Cas9 system to dystrophic muscle or satellite cells in vivo. The AAV delivery system has proven to be safe and effective and has already been advanced in clinical trials for gene therapy¹. In addition, the AAV9 serotype has been shown to provide robust expression in skeletal muscle, heart, and brain, the major tissues affected in DMD patients.

Another challenge with respect to the feasibility of clinical application of the CRISPR/Cas9 system is the increase in body size between rodents and humans, requiring substantial scale-up. More efficient genome editing in postnatal somatic tissues is also needed for the advancement of the CRISPR/Cas9 system into clinical use. Although CRISPR/Cas9 can effectively generate non-homologous end joining (NHEJ)-mediated indel (insertion or deletion) mutations in somatic cells, homology directed repair is relatively ineffective in postmitotic cells. That is to say, cells that do not exhibit mitosis and cell division after fetal development is complete (such as myofibers and cardiomyocytes) because these cells lack the proteins essential for homologous recombination².

So, although it might be possible in future for the CRISPR/Cas9 system to enhance homology directed gene repair, safety issues of the CRISPR/Cas9 system, especially for long-term use, need to be evaluated in preclinical studies in large-animal models of disease. Despite the challenges, with rapid technological advances of gene delivery systems and improvements to the CRISPR/Cas9 editing system there is light ahead for those dedicated to finding new therapies.

A new hope There was agreement between the three laboratories on restoration of dystrophin-positive fibres. Significant functional recovery was demonstrated in CRISPR/Cas9-treated mice which showed improvements including increased grip strength, improved force generation, resistance against eccentric contraction, and reduced serum creatine kinase (CK). Histologically, there was reversal of muscle necrosis, fewer infiltrating inflammatory cells, and decreased fibrosis. There are many outstanding legal wrangles and battles over who gets the glory for CRISPR and its priceless intellectual property rights (see here) but fortunately they have so far not stalled these frontline studies using the CRIPSR techniques and variants. They offer some hope at last for families of victims of a heartbreaking condition.

Author: Dermot Martin is a science journalist with a special interest in the life sciences and techniques for analytical chemistry.

References: 1. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987;50:509–17 2. Inhibition of non-homologous end joining increases the efficiency of CRISPR/Cas9-mediated precise genome editing Maruyama, Dougan, Truttmann et al