The ability to edit an organism’s genome using CRISPR has provided myriad applications ranging from crop development to novel therapeutics, while editing of various animal species offers hope in areas from de-extinction to xenotransplantation. But what about the domestic cat?
In a new report published in The CRISPR Journal, researchers at InBio, a biotech company in Virginia (formerly known as Indoor Biotechnologies) specializing in products and services in environmental sciences, allergy and asthma, have applied CRISPR in research designed to reduce human allergies to our favorite feline friends.
The domestic cat is the most common source of mammalian allergen, with cat allergies affecting up to 15% of adults and children. While common treatments (e.g. antihistamines) are limited to addressing the allergic symptoms, InBio has sought to tackle allergies at the cat source.
Fel d 1, a protein produced by cat salivary and sebaceous glands, is the main allergen culprit. It has been documented to mediate the allergy response in 95% of cat allergy patients. Structurally, Fel d 1 is a tetrameric protein composed of two heterodimers, each of which consists of two chains, chain 1 and chain 2, coded by the genes CH1 and CH2 respectively. Led by Nicole Brackett, InBio’s approach used sequence and structural analysis of Fel d 1to identify conserved coding regions suitable for CRISPR editing.
Brackett’s team applied phylogenetic analyses across twenty-four exotic cat genomes representing eight big or wild cat species and found that Fel d 1 gene sequences lacked evolutionary conservation. These results implied that these genes are non-essential for cats and thus would be suitable targets for deletion. The biological function of Fel d 1 remains unknown.
The authors went on to show that Fel d 1 in immortalized feline cells is amenable to gene editing using CRISPR, with editing efficiencies of up to 55% and no evidence of editing at predicted potential off-target sites. These results provide the first step in creating Fel d 1 knockout cats.
“Current treatments for cat allergic disease merely reduce allergic symptoms or have shown limited or inconsistent efficacy. Several new approaches aim to neutralize or reduce exposure to the allergen, however modest reductions in allergen levels may be clinically insufficient,” stated Brackett. “Our approach of targeting the allergen with CRISPR technology would be the first treatment option to effectively remove the major cat allergen from the source, which will be a significant advance compared to existing treatment options.”
The authors suggest additional studies are needed before transitioning to CRISPR knockouts in vivo in cats. For example, InBio’s current approach evaluated the editing efficiencies of targeting to either CH1 or CH2 individually. Future studies will investigate the efficiency of simultaneous knockouts of the Fel d 1 genes using multiple targets. In addition, off-target analysis will be expanded to identify genome-wide double strand breaks using screening approaches, such as GUIDE-seq or CIRCLE-seq to further elucidate the effects of the knockout in cells. These CRISPR knockout studies will also be replicated in Fel d 1-expressing primary feline cells.
Feline lucky
As described by Sarah Zhang in The Atlantic, applying biotechnology to tackle cat allergies is not a new feat. From immunity transfer using egg-yolk coating on Purina’s cat food to cat vaccines that reduce shedding of Fel d 1, the goal of a true hypoallergenic cat remains on the industry’s radar. Is CRISPR genetic therapy next on the list?
In an upcoming commentary for GEN Biotechnology, Alison Van Eenennaam, professor of cooperative extension in animal biotechnology at the University of California, Davis, ponders the regulatory path for future CRISPR-enabled hypoallergenic cats. She writes that, “given cats are not food animals (at least in the United States) and assuming that knocking out Fel d 1 poses no ill-effects to the cat itself, the question would be whether such cats pose a safety concern to humans or the environment.”
Brackett states that InBio anticipates developing a Fel d 1 knockout cat as a proof-of-principle, but does not intent to create and subsequently breed Fel d 1-free cats.
“From a consumer/patient perspective, KO cats would be largely cost-prohibitive. We also think it would be more practical from a commercial standpoint, as well as more ethical, to develop a treatment that is administered to existing cats rather than breeding and selling allergen-free cats,” Brackett said.
“At this moment, we envision [cat owners or their vets injecting their pets periodically to shut down expression of Fel d 1], though the success of this treatment will depend on the development of suitable delivery mechanisms to specifically target Fel d 1-expressing cells and tissues with the requisite CRISPR reagents, and potentially the age of the cat,” Brackett continued.
