The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease-9) system has been a model of great promise in the gene therapy research community since 2012. It was initially discovered as the adaptive immune system of certain bacteria and archaea. In this paired system, the Cas9 nuclease can target and cleave foreign DNA (1).

Since its discovery, the targeted sequence “cutting” ability of Cas9 has been proposed as a method of genome editing. It was found that when complexed with a short guide RNA (gRNA), the Cas9 nuclease can be used for site-specific genome cleavage, which can result in nucleotide insertions and/or deletions or allow for donor DNA to produce replacement mutations. Additionally, a defective, nuclease-deficient form of Cas9 can be utilized for targeted gene silencing or activation (1, 2). The capabilities of this new gene editing technique, and the progress made in vivo in rodent models, created excitement among researchers as a potential therapy for various human illnesses, such as sickle cell disease, muscular dystrophy, and retinitis pigmentosa (3, 4).

As research into Cas9/gRNA as a gene therapy has progressed, however, some associated risks have been identified, adding hurdles on its journey to the clinic (4). One of the most pressing issues, which has been recently highlighted in a study (not yet peer-reviewed) performed by Carsten T. Charlesworth et al. and a comprehensive review by Wei Leong Chew et al., is the immunogenicity of the Cas9 nuclease. The Charlesworth study explained that most commonly used homologs of Cas9 are derived from Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes), two bacteria to which most humans have pre-existing antibodies, due to the frequency at which they infect the population. The study predicted that if pre-existing adaptive immunity to these two bacteria is found in most of the human population, then both the S. aureus and the S. pyogenes homologs of Cas9 (SaCas9 & SpCas9) would be recognizable antigens as well (3).

The study found the prediction of immunogenic potential to be true. In fact, 79% of donors produced anti-SaCas9 antibodies, and 65% of donors produced anti-SpCas9 antibodies. This data raised awareness to the fact that use of the Cas9/gRNA system as a therapeutic may not be safe and effective as it exists now (3). Though this news might discourage many, it excites the scientists at EpiVax, who are always up for a challenge. Our team specializes in immunogenicity risk assessment…but even better, we are experts in protein deimmunization and tolerization. We’ve successfully re-engineered protein therapeutics (like FVIII, alpha interferon, and Botox) before, and we could do it again with Cas9!

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