The CRISPR/Cas9 genome editor and its therapeutic potential

In the first few weeks of the quarter we had a lengthy discussion on the origins of cancer cells. Loss of function mutations in tumor suppressor genes and gain of function mutations in oncogenes lead to the deregulation of the cell cycle and eventually uncontrolled cell proliferation. Gene therapy has been considered as an option to rehabilitate these genes back to a functional state. Unfortunately, the methods researched in the last three decades since the principle discovery of gene therapy, such as retroviral vectors, have been incredibly inefficient and have a low ceiling due to inherent low indel (nucleotide insertions or deletions) specificity. Within the last fifteen years though, three restriction enzymes (used to cut DNA at relatively specific sites) have been heavily researched in hopes of finding a mechanism to make the ultramodern form of gene therapy a reality.

The first of three enzymes discovered, the Zinc-finger nuclease (ZFN) has been reengineered and researched so heavily in the last decade that a couple of months ago a paper was published in the NEJM  (Kay et al. 2014) outlining how ZFNs were used to introduce a 32bp deletion in the CCR5 gene of an HIV patient as a way of introducing HIV-resistance in the patient’s T-cells. TALENs, the second endonuclease, has proven to be a much easier enzyme to work with and appears to have a higher ceiling than ZFNs due to enhanced sequence specificity, but has not reached the level that ZFNs have in being used as a gene therapeutic. The newest, and by far the most exciting, of the three enzymes is known as the CRISPR/Cas9 endonuclease system. This endonuclease, discovered in the bacteria responsible for the fermentation of dairy, Streptococcus thermophilus, is used as a method of protecting the bacteria from viral infection by explicitly targeting viral DNA in the bacterial cytosol and introducing double strand breaks in the viral genome, thereby halting viral reproduction. This enzyme has two notable domains to focus on, a domain that cuts DNA, and a single guide RNA (sgRNA) domain that guides the CRISPR/Cas9 enzyme to the matching viral sequences. This guiding RNA in the CRISPR/Cas9 is what makes it such an exciting discovery that will inevitably affect all fields of biology. When engineering the CRISPR/Cas9 endonuclease to cut a specific sequence, all that needs to be altered is the short (20bp) RNA sequence in the protein, much easier than altering the protein structure of both ZFNs and TALENs. This versatility of CRISPR/Cas9 allows libraries of the enzyme to be engineered cheaply and opens up the future of gene therapy to masses. One of the head researchers involved in discovering the system has publicly praised her enzyme by stating that the CRISPR/Cas9-system “could potentially be as powerful as the Polymerase Chain Reaction (PCR).”

Without going into detail, the combined CRISPR/Cas9-system was discovered in 2007 and in the last seven years, the “CRISPR Craze” (Figure 1) has lead to scientists flocking to study this system. With only seven years since its discovery, CRISPR/Cas9 is far from being used in human therapy. Current research is focused on removing some of the limitations of the enzyme and proving that the enzyme has functionality in eukaryotic cells. 

Figure 1. CRISPR Craze. Dramatic increase in CRISPR
related research since 2007, with an estimated 314 publications in
2014 (Source: Web of Science, March 17, 2014).

Interestingly though, a recent paper (Yin et al. 2014) claims that it is the proof of principle that the CRISPR/Cas9 endonuclease can be utilized as a therapeutic in vivo. To investigate the efficacy of CRISPR/Cas9 in a mammalian system the researchers studied a mouse model with a fatal mutation in the fumarylacetoacetate hydrolase (Fah^mut/mut), the last enzyme in the tyrosine catabolic pathway. Fah^mut/mut have a mutation in exon 8 of the gene (G—>A) that results in the skipping of the exon and an detrimentally truncated protein. To fix the resulting lethal phenotype, severe liver damage, the mice were injected with a plasmid vector expressing the Cas9 enzyme and one of three sgRNA targeting the Fah locus (FAH1, FAH2, or FAH3), and a high concentration of ssDNA containing the wild type Fah gene, which can serve as a template for the DNA repair process once CRISPR/Cas9 cuts the mutant gene. 

Figure 2 shows that although the phenotype was improved, relative to the control, a fraction of the cells in the FAH2 treated group were saved. The paper states that only ~0.4% of hepatocytes responded to the treatment, possibly due to the vector used or indirect injection of treatment. However, the extent of the damage, as shown in figure 3, is clearly enhanced to a significant extent. Demonstrating that in treated cells, the phenotype is functionally rescued to the level of control NTBC treated mice. Finally, figure 4 illustrates that treated cells show an 8-36% level of expression relative to WT mice, which again shows that this article is landmark only for the sake of proof of principle, not efficacy. 

Figure 2. Histological stain of mouse 

hepatocytes show improved phenotype in 

Cas9 + sgRNA treated cells (FAH2). 

Wild-type mice (Fah^+/+), control mice 

(Fah^mut/mut + Unguided Cas9), Cas9 + sgRNA 

treated mice (Fah^mut/mut + FAH2).

Figure 4. Quantitative-PCR measurement of wild-type
mRNA (G allele) expression in wild-type mice (WT), Fah^mut/mut mice (Mut), and in treated mice
(FAH1, FAH2, and FAH3). 

Figure 3. Liver damage marker (aspartate 

aminotransferase (AST), alanine aminotransferase 

(ALT), and bilirubin) levels show over three-fold 

decline in treated mice. NTBC is an upstream inhibitor 

of the tyrosine catabolic pathway that alleviates the Fah^mut/mut 

phenotype; "NTBC on" mice are liver damage control group 

within the Fah^mut/mut genotype, "NTBC off" are untreated 

mice with full extent of lethal phenotype; "NTBC off + FAH2" 

are treated mice. * P < 0.01, n = 3 mice.

Relative to the hereditary tyrosinemia in these mice, most cancers are much more heterogeneous (read: more complicated) leading one to think that the methods from taking this therapy from reaching 0.4% cells and expressing 8-36% WT-mRNA in said affected cells to an efficacious standard in cancer patients are far from being reached. However, the CRISPR/Cas9-system has excited thousands of scientists around the world for many reasons and research will keep advancing on the system in the coming years at a dramatic rate. Currently, though, the value of the system as a gene therapeutic in humans can only be measured in its potential and not in its capability.

References

1. Yin, H., Xue, W., Chen, S., Bogorad, R. L., Benedetti, E., Grompe, M., et al. (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnology. doi:10.1038/nbt.2884
2. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096), 816–821. doi:10.1126/science.1225829
3. Kay, M. A., & Walker, B. D. (2014). Engineering Cellular Resistance to HIV. New England Journal of Medicine, 370(10), 968–969. doi:10.1056/NEJMe1400593