Negative Autoregulation for Safe Cas13-Based RNA Therapeutics

Mitigates Collateral RNA Damage in Repeat Expansion Disorders

These negative autoregulatory approaches enable safe Cas13-based RNA therapeutics for mitigating collateral RNA damage when treating repeat expansion disorders. CRISPR-Cas13d is an enzyme in the CRISPR family of genome editing tools. Cas13d has been shown to have immense therapeutic potential for treating RNA mediated diseases like myotonic dystrophy type I, or other repeat expansion diseases, affecting 1 in 3000 people worldwide . However, Cas13d is highly susceptible to non-specific RNA cleavage, or the accidental cleavage of the wrong section of RNA. These types of erroneous events can cause collateral damage in the targeted cells, creating significant safety risks to therapeutic use in humans. Therefore, there is an evident need for methods to reduce collateral damage and improve the safety of Cas13-based therapeutic applications.

Researchers at the University of Florida have developed negative autoregulatory approaches for safe Cas13-based RNA therapeutics. This mitigates collateral RNA damage experienced in the targeted cells while isolating the therapeutic benefits of the treatment. Both methods aim to limit the expression of Cas13d in the cells while improving safety and maintaining the actual benefits associated with the treatment. This technology provides a solution for an extremely limiting factor in the use of Cas13d for repeat expansion disorders, potentially opening the door to this enzyme’s use without needing to consider negative side effects. This platform has the possibility to reimagine the genetic therapeutics market.

Application

Enables safe Cas13-based RNA therapeutics through two negative autoregulatory approaches, mitigating collateral RNA damage when treating repeat expansion disorders

Advantages

• Limits Cas13d expression, reducing collateral cell damage and improving therapeutic safety
• Destroys pathogenic CUG RNA, providing effective treatment for diseases like myotonia, muscle atrophy, hypersomnolence, cataracts, and cardiac arrhythmia
• Enables compact AAV packaging, allowing in-vivo delivery of the therapeutic construct
• Allows interchangeable regulatory strategies, creating research opportunities to tailor expression control for each method's use

Technology

These two negative autoregulatory platforms enable safe Cas13-based RNA therapeutics by mitigating collateral RNA damage when treating repeat expansion disorders. Both platforms aim to limit Cas13d expression, thus minimizing off target RNA splicing and in turn, reducing collateral damage. The first method uses the KRAB repression system as well as zinc finger fusion to negatively regulate expression of the enzyme. The second method uses internal knockdown to limit the enzymes' expression. With these two platforms, the risks associated with collateral damage are mitigated while the therapeutic benefits of the enzyme are maintained. Therefore, these methods can render Cas13d a safer and more effective treatment than ever before.