Reports of developments in CRISPR-Cas9 technologies are near-ubiquitous, with scientific and mainstream news outlets alike regularly announcing breakthroughs facilitated by the technology. However, despite all the potential applications for CRISPR-Cas9 gene editing, it is perhaps those that concern personal health that have captured the public’s imagination.
CRISPR-Cas9 editing has obvious applications in diseases with a clear genetic basis, especially in monogenetic disorders where the condition is caused by a single gene mutation. Though not universally the case, many cancers are caused by such mutations. Projected to affect nearly half the population over the coming decades, cancer arises from deleterious mutations in the cell that allow the cell to divide uncontrollably. The uncontrolled proliferation can lead to aggregates of cells – tumours – that may inhibit other bodily functions by sequestering nutrients or blocking pathways. Individual cells may break away from these tumours and metastasise to other parts of the body, creating more tumours.
However, the nature of the mutations that lead to cancer are increasingly well understood. With this understanding comes the ability to offer “personalised molecular surgical therapy” through CRISPR-mediated gene editing (Tang and Shrager, 2016).
Various proof-of-principle experiments have been conducted to show that CRISPR-Cas9 can correct heredity diseases in vivo. For example, CRISPR-Cas9 successfully induced wild-type FAH protein expression in mice with hereditary tyrosinemia type 1 (HTI), which results in impaired tyrosine metabolism (Yin et al., 2014). However, it is possible that the presence of the CRISPR-Cas9 system will elicit an immune response in cells expressing it as it is a foreign (bacterial) protein, leading to a potentially damaging immune response (Chew et al., 2016).
Steps have already been taken to transition the gene-editing technology from laboratory to clinic. In October 2016, a Chinese team announced they had introduced modified T-cells into a patient suffering from an aggressive form of lung cancer (Cyranoski, 2016). CRISPR-Cas9 had been used to modify PD-1, a cell-surface receptor usually expressed on T-cells that dampens the immune response. Thus, if present on cancer cells, it prevents them being attacked and cleared by immune cells. Knocking out this gene would allow a full immune response against the tumour cells to be mounted.
There are, of course, other diseases that could also be cured by CRISPR-Cas9 editing. Many past gene therapies rely on the integration of new genes into the patient’s genome using viral vectors such as adeno-associated viruses (AAVs) or lentiviruses. Though it depends on the vector, this method runs a high risk of insertional mutagenesis, whereby recombinant DNA introduced as part of the gene therapy disrupts an important gene or regulatory region. Such a result was seen in a recent X-linked chronic granulomatois disease (X-CGD), where one boy developed myelodysplastic syndrome (MDS) due to insertional activation of EVI1 and STAT3 genes (Siler et al., 2015). As CRISPR-Cas9 is more precise than any other gene-editing technology to date, such incidents may be reduced.
Human medicine may also benefit from the modification of other animals’ genomes. Xenotransplantation is a developing therapeutic technique where tissues or organs from one species are transplanted into a recipient of a different species. With a universal and chronic shortage of human donor organs, if the procedure proved successful it would reduce the number of patients who suffer severe morbidity or even, unfortunately, die whilst on a waiting list.
There are, however, many factors that prevent the immediate implementation of xenotransplantation. The high disparity between interspecific cell-surface molecules means that the graft will usually be attacked by the recipient’s immune system, resulting graft failure. However, there are other xenoreactive natural antibodies (XNAs) that could also induce rapid (or “hyperacute”) rejection (Goodman, Pearse and d’Apice, 1998). In pigs (whose size and physiology make them ideal candidates to donate to humans) the α-galactosyl moiety on cells is highly immunogenic once introduced to humans, eliciting a hyper acute response. However, if pigs can be edited with CRISPR-Cas9 to knock out the 1,3 galactosyl transferase gene, the carbohydrate won’t be expressed and thus won’t cause an immune response.
Perhaps even more obscurely, many allergy suffers may benefit from the CRISPR revolution. Egg allergies, for example, have a global incidence of approximately 2% in children (Reardon, 2016) and is usually an aberrant immune response to either ovalbumin or ovomucoid in the albumin (Oishi, 2015). However, if chickens can be edited such that these proteins are not expressed, the allergic reaction would be avoided. As well as allowing suffers to eat a broader variety of foods, they would also be able to receive many routine vaccines that are manufactured using chicken eggs.
Despite its precision, editing with CRISPR-Cas9 may result in off-target effects (Wu et al, 2014), where areas of the genome that are very similar in sequence to the desired target are unintentionally targeted by the CRISPR guide and cleaved. Newer CRISPR-Cas9 variants reportedly reduce off-target cleavages (e.g. Kleinstiver et al., 2016; Tabebordbar et al., 2016). Given thepotentially devastating nature of off-target cleavages in therapeutic applications, the reliability of these methods must be intensely studied and have high replicability in animal models before they are transferred to humans.
Whilst these improvements alleviate some safety problems, others remain: in clinical settings, issues of low efficacy still threaten patient wellbeing (Dai et al., 2016) and delivery mechanisms may elicit strong and damaging immune responses (Li et al., 2015). There is the additional aforementioned concern that the allogenic nature of the system will elicit an immune response inthe recipient.
The efficiency, relative ease of use and speed of CRISPR-Cas9 has led to its rapid dissemination in laboratories as the foremost method to edit genomes. It offers very real therapeutic potential for many conditions that thus far have lacked treatments whilst broadening the range of treatments available for other conditions. There are, of course, hurdles that remain, but that rateat which the science of genome editing means that clinical trials have already begun, bringing the prospect of “molecular surgeries” closer to patients than ever.
Chew, W.L., Tabebordbar, M., Cheng, J. K.W., Mali, P., Wu, E.Y., Ng, A.H.M., Zhu, K., Wagers, A. J. and Church, G.M. (2016). A multifunctional AAV-CRISPR-Cas9 and its host response Nature Methods 13: 868-874 doi:10.1038/nmeth.3993
Cyranoski, D. (2016a). Chinese scientists to pioneer first human CRISPR Nature 535: 476-477 doi:10.1038/nature.2016.20302
Dai, W-J., Zhu, L-Y., Yan, Z-Y., Xu, Y., Wang, Q-L. and Lu, X-J. (2016). CRISPR-Cas9 for in vivo gene therapy: promise and hurdles. Molecular Therapy Nucleic Acids 5: e349 http://dx.doi.org/10.1038/mtna.2016.58
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Siler, U., Paruzynski, A., Holtgreve-Grez, H., Kuzmenko, E. et al. (2015). Successful combination of sequential gene therapy and rescue allo-HSCT in two children with X-CGD – importance of timing. Current Gene Therapy 15: 416-427
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Tang, H. and Shraber, J.B. (2016). CRISPR/Cas-mediated genome editing to treat EGFR-mutant lung cancer: a personalized molecular surgical therapy. EMBO Molecular Medicine DOI 10.15252/emmm.201506006
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