Study demonstrates potential of novel mutant enzyme in treatment of sickle cell disease

A study, recently published in Nature Medicine, has demonstrated that a novel mutant enzyme may offer potential for medical use in the treatment of sickle cell disease (SCD).

Researchers at Integrated DNA Technologies (IDT) and the laboratory of Professor Matthew Porteus at Stanford University have described a novel Cas9 mutant that shows improved specificity and maintains high activity when used in the medically relevant ribonucleoprotein (RNP) format.

Additionally, the researchers demonstrated its potential for medical use in human haematopoietic stem progenitor cells (HSPCs), where the mutant enzyme was able to correct the mutation in the beta-haemoglobin gene responsible for SCD.

Other groups have described Cas9 mutants with improved specificity, however, in every instance these have shown reduced activity when used in the clinically relevant RNP format. These previous attempts were developed with an intelligent design based on known protein crystal structures.

In this latest work, the IDT scientists used an unbiased method to screen approximately 250,000 random Cas9 mutants to identify those rare mutants that improved specificity without compromising activity. After several rounds of selection, a single mutant emerged, now known as Alt-R HiFi Cas9 nuclease, that provides the desired high on-target, low off-target characteristics. Then in collaboration with Stanford, the researchers were able to demonstrate the robust on-target editing and minimal off-target cleavage achieved by HiFi Cas9 in several therapeutically relevant loci in hard-to-edit HSPCs. They also showed HiFi Cas9-mediated correction of the sickle cell disease-causing p.E6V mutation in patient-derived HSPCs.

“Previous attempts at improving Cas9 specificity characterised the mutants using plasmid-based methods that result in sustained overexpression of the Cas9 protein, which increases off-target activity and is not ideal for medical applications,” explained Dr Mark Behlke, PhD, chief scientific officer at IDT and a co-author of the study. “This sustained overexpression, however, rescued function of the mutants that otherwise showed low activity when used in the more transient RNP format. We specifically performed a broad screen to identify a mutant that performs well when used at the lower protein levels achieved with RNP delivery, maximising safety and further reducing unwanted side effects. Prof. Porteus demonstrated utility using the new system to correct the SCD mutation in normal human blood-forming stem cells while minimising known off-target activity. We anticipate significant interest in use of the new Cas9 mutant in translational medical applications.”