CRISPR/Cas9-HDR Gene Therapy To Treat OCA1 Albinism

Abstract

Gene therapy has gained large interest in the scientific field for potential therapeutic applications since the development of CRISPR/Cas9 genome editing techniques. The effectiveness of CRISPR/Cas9 to treat disease has been demonstrated in studies regarding HIV, liver disease, and triple negative breast cancer, but little is known about its ability to treat oculocutaneous (OCA1) albinism. OCA1 albinism is associated with mutation in the tyrosinase enzyme gene ( ​TYR​), and is characterized by little to no melanin synthesis, particularly in hair follicles, skin, and eye tissues. We propose the use of hydrodynamic tail vein injections of wildtype ​TYR ​ssDNA with guide RNA and cas9, as per CRISPR/Cas9-HDR gene therapy techniques, to increase melanin synthesis in B6 albino mice. Melanin quantities will be assessed after mouse sacrifice at 4 months of age by collecting skin, eye tissue, and hair follicles and then quantifying melanin using spectrophotometric techniques. Melanin quantities will then be compared to a negative control cohort. If this gene therapy is successful in increasing melanin synthesis, CRISPR/Cas9-HDR could prove a promising field in OCA1 albinism treatment.

Introduction and Scientific Background

Oculocutaenous albinism (OCA) is an inherited, autosomal recessive genetic disorder Nthat is characterized by a reduction or lack of melanin pigment (NORD, 2019). There are seven forms of this disorder, caused by mutations in seven different genes that control melanocyte production (NORD, 2019). In specific, oculocutaneous albinism 1 (OCA1) is associated with mutations in the tyrosinase ( ​TYR​) gene, and consists of 2 subtypes; OCA1A and OCA1B, characterized by no melanin synthesis and minimal melanin synthesis, respectively (NORD, 2019).

The TYR gene produces the tyrosine rate-limiting enzyme ​ found in melanocytes, and plays a major role in the production rate of melanin (Lewis, 2000). OCA1A is caused by null variants in TYR leading to an inactive form of tyrosinase enzyme polypeptide that blocks the first step of the melanin biosynthetic pathway. OCA1B is caused by pathogenic variants in TYR, producing a truncated tyrosinase enzyme (Lewis, 2000). OCA1 affects approximately one in 40,000 globally, and OCA1A is dominant over OCA1B (NORD, 2019). CRISPR/Cas9 is an advanced genome editing technology that is revolutionizing diverse fields of biotechnology and medical research (Adli, 2018). The system consists of 2 important molecules that can introduce mutation into DNA: the cas 9 enzyme — molecular scissors that can alter DNA by cutting the genome at any desired place, and guide RNA (gRNA) — a small pre-designed RNA sequence about 20 bases long, located within a longer RNA scaffold. CRISPR/Cas9 may be used to mediate homology directed repair (CRISPR/Cas9-HDR), where a donor single stranded DNA (ssDNA) template is inserted into the location cut by cas9. Natural DNA-repairing mechanisms will aid in binding the ssDNA, ultimately allowing for high-precision genetic manipulation ​ (Your Genome, 2016).

There has been a multitude of research in the past regarding uses of CRISPR/Cas9 in curing or preventing genetic disorders. However, there have not yet been any studies conducted on its use in OCA1 albinism. This report seeks to determine whether CRISPR/Cas9-HDR can be used as a tool in manipulating the mutated ​TYR​ gene responsible for OCA1, in hopes of providing a viable treatment for this genetic disease.

Research Hypothesis and Rationale

While CRISPR/Cas9 gene therapy to correct OCA1 albinism has been explored when altering known mutated gamete cells (Song et al., 2018), little is known about the ability of CRISPR/Cas9-HDR for in vivo treatment of ​TYR​ mutated somatic cells. To evaluate if using CRISPR/Cas9-HDR to replace mutated ​TYR​ genes can increase melanin production in OCA1 albinism, we propose a preliminary test that explores this gene editing technique in B6 Albino mice. Should CRISPR/Cas9-HDR be effective in replacing mutated ​TYR​ genes, mice should show increased melanin in affected tissues, such as the skin, iris, retina, and hair follicles, as compared to negative controls.

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The effectiveness of CRISPR/Cas9 for gene therapy has been demonstrated in various contexts. For example, a study published in 2013 determined that CRISPR/Cas9 has the potential to disrupt HIV-1 genome expression in cells, proving that CRISPR/Cas9 may be useful in the treatment of HIV infection (Ebina et al., 2013). CRISPR/Cas9 has also been used to perform genome editing in the adult mouse liver, through hydrodynamic tail vein injection (Niola et al., 2019). If CRISPR/Cas9-HDR gene therapy techniques are successful in replacing mutated ​TYR​, it could prove a promising clinical treatment for individuals with OCA1 albinism.

Research Methodology

To study the effects of proposed CRISPR/Cas9-HDR replacement of ​TYR ​in stimulating albinism-suppressed melanin production, a B6(Cg)- ​Tyr​c-2J​/J (B6 albino) mouse model will be used, available for purchase through The Jackson Laboratory (The Jackson Laboratory, 2019). As this mouse line is simply a common C57BL/6J black fur mouse with a tyrosinase gene mutation, the positive control cohort will consist of unmutated C57BL/6J mice. Similarly, the negative control cohort will consist of untreated B6 albino mice. The dependent variable is the level of melanin in tissues, and the independent variable will be the presence of the unmutated TYR​ or mutated ​TYR​ gene.

Upon weaning, 20 B6 albino mice will be subjected to gene therapy using hydrodynamic tail vein injections of tyrosinase ssDNA and synthesized gRNA targeting the mutated ​TYR​ gene sequence. Studies by Niola et al. and Zhu et al. confirmed this an effective technique in manipulating somatic cell genes with measurable changes manifesting as early as three weeks post-injection (Niola et al., 2019). Positive and negative control cohorts will not receive gene therapy injections.

To confirm successful transformation of somatic cells in the experimental group, mouse DNA samples will be collected four weeks post injection, and tested for the presence of the unmutated ​TYR​ gene using PCR and gel electrophoresis techniques. Similar testing will be done in positive control mice, to confirm for unmutated ​TYR​ gene presence, and negative control mice, to confirm for mutated ​TYR​ gene. While there should manifestations of melanin production in experimental mice, such as gaining coat pigment, we will quantify melanin production upon sacrificing mice at 12 weeks of age. Skin, eye tissue, and hair follicle samples will be harvested and analyzed for melanin using spectrophotometric techniques, outlined in Watt et al.’s paper regarding melanin testing of hamster tissues (Watts et al.,1981). These data will be subsequently compared to similarly collected data from positive and negative control cohorts. If B6 albino mice treated with gene therapy present a statistically significant higher melanin quantity in tissues than untreated negative controls, we can conclude that CRISPR/Cas9 mediated HDR may be a viable treatment for stimulating melanin production in OCA1 albinism.

Conclusion

It is evident that further research and clinical trials must be conducted to review the safety, and eliminate off-target effects of this procedure, until it may be routinely used to treat disease in humans. Ultimately, this study could provide an important base of preliminary research into the use of CRISPR/Cas9 to eliminate genetic diseases in the future.

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