The challenges of translating CRISPR to the clinic

CRISPR gene editing

CRISPR-Cas9 is revolutionizing all facets of drug discovery, allowing for both a deeper understanding of disease processes, and for the development of ground-breaking genetic medicines. 

As these new classes of cell and gene therapies translate to the clinical setting, some key bottlenecks remain to be solved before the technology will realize its full societal impact.

Dr Alasdair Russell, CSO of Zygosity, highlights where the field is at with some of the most talked about bottlenecks, namely those involving the safety of CRISPR technologies.

Safety is often the first port of call when considering the therapeutic application of CRISPR technology. Where are we with unintended, off-target editing away from our target gene?

CRISPR-Cas9 is an incredibly precise and efficient tool, however it can be prone to rare, unintended editing away from the target gene. While it is true that CRISPR can edit at unintended sites of the genome, raising valid safety concerns, there are now a swathe of computational tools and assays available to assess and quantify these events. 

Indeed, in March this year the FDA released draft guidance on the use of CRISPR in gene therapy products. The FDA recommends characterization of on- and off-target editing using multiple orthogonal methods (e.g. in silico, biochemical or cell-based assays) and across multiple patient samples (where possible). 

With this framework in mind, leading CRISPR therapy companies have published their off-target strategies in an effort to align the field in a spirit of openness, giving a clear roadmap for future CRISPR therapy development.

More recently, several groups have shown that there are unintended edits at the target site, including high profile cases involving DNA shuffling. How is the field coping with this finding?

CRISPR-Cas9 works by cutting DNA within a gene and allowing error-prone DNA repair to introduce errors in the hope that these will silence the target gene. DNA repair in the cell is probabilistic, in that a given DNA cut can be repaired in any number of different ways.

What this looks like when applied to a group of cells (e.g. T-cells for a CAR-T therapy) or an intact tissue (e.g. retinal tissue in the eye) is that different cells will contain different edits – and not all of them will silence the target gene. This is a type of genetic impurity (called mosaicism) and is pervasive across all species. 

Currently there are no viable solutions to solve genetic impurity at the target site for cell and gene therapy.

Other, less common, but still undesirable events affecting the target gene include large deletions and genomic rearrangements. Several academic groups have observed rare loss of long stretches of DNA at the target site after prolonged exposure to CRISPR-Cas9. Further, when multiple genes are targeted for silencing simultaneously, it is possible for intervening segments of the genome to shuffle. 

This phenomenon, at least in part, has contributed to Beam Therapeutics’ FDA hold on the BEAM-201 base editor. In response to this hold, the field has adopted a more rigorous approach to mapping the frequency of any genome shuffling in a manner akin to how off-target editing is now being approached. Indeed, the draft guidance by the FDA recommends an assessment of genome shuffling and large deletions by robust methodologies.

Immuno-oncology medicines (e.g. CAR-T therapies) have been a revolution in the treatment of blood cancers. The next generation of these medicines use CRISPR to layer on enhancements (better survivability, more efficacy, less exhaustion). Will these next generation of CRISPR CAR-T medicines allow us to finally target solid tumors?

The overwhelming majority of CRISPR therapy companies have at least one program focused on next generation cell therapies. To enhance these therapies (persistence, efficacy, safety) multiple genes need to be silenced within a given cell. Multiplex silencing represents a significant roadblock to CRISPR realizing its full potential in this space. 

It is now recognized that between three and 12 gene modifications are necessary to enhance autologous and allogeneic CAR-T therapies respectively. As described above, CRISPR results in genetic impurity at the target gene, with only a fraction of edits silencing the gene. Importantly, as you scale the number of genes to be silenced, you drastically reduce the probability that you will get a cell that contains all the desired edits in it. 

As an exemplar, there are published cases where triple-gene silenced next generation CAR-T therapies administered to patients contained less than 2% of cells with the desired edit across all three genes. The low proportion of multiplexed edited cells presents an enormous manufacturing challenge as investigators have to invest heavily in novel methods to purify out these rare, therapeutically relevant cells.

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In some settings this presents an enormous hurdle, for example autologous cell therapies where the patient-derived T-cells themselves which are used to make the CAR-T medicine are compromised. This is due to the T-cells being derived from patients who have received a toxic preconditioning chemotherapy regimen. In this setting, multiplex gene editing to generate next generation CAR-T medicines is generally considered infeasible.

To get around this issue, the majority of CAR-T programs focus on using healthy donor T-cells as a base upon which to develop off-the-shelf CAR-T medicines. Here the challenge is that often substantially more genes (up to 8-12) may need to be silenced to overcome rejection due to the CAR-T’s being seen as foreign (graft vs host disease). 

This presents the concept of the ‘multiplex editing ceiling’, above which it is unfeasible to silence more genes (due to genetic impurity). Currently the multiplex ceiling falls significantly short of the 8-12 genes needed to be silenced simultaneously. This multiplex ceiling driven by DNA impurity at the cut site will need to be overcome if the next generation CAR-Ts are going to help the market expand from $1.2 billion (2021) to $22 billion in 2031 as predicted.

What are the future challenges that we will face in the therapeutic application of CRISPR-Cas9?

Looking forward, as we move to treat more and more genetic disease within the body, we will need to grapple with genetic impurity. Within an intact diseased organ it is impossible to apply a manufacturing process to ‘purify out’ cells with desirable edits. Until this is solved, only diseases with broad therapeutic windows (i.e. small levels of gene correction or silencing) will be accessible to most CRISPR technology.

Further, a less appreciated bottleneck is the fact that the vast majority of cells in our bodies which are affected by genetic disease are not undergoing active cell division. This is an important observation as all current technologies for rewriting genes (including base editors and PRIME editors) are inefficient in cells that are not cycling. We as a field will have to reconcile this if CRISPR is truly to revolutionize medicine.

Zygosity is a proprietary genome editing platform that develops accurate and precise CRISPR medicines that are not burdened by impure edits and unpredictable phenotypes.


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