The Need

There are thousands of monogenic diseases that are lacking effective therapeutic treatments. The main bottleneck in developing effective treatment is the ability to effectively edit gene while controlling for the myriad of off-target effects that come with targeting the genome. Duchenne Muscular Dystrophy (DMD) is once such monogenic disease that is caused by genetic mutations in the DMD gene, which encodes the dystrophin protein. The mutations including missense point mutations, nonsense point mutations, deletions, and insertions lead to the disruption of dystrophin protein translation. Correction of such mutations or restoration of the reading frame by skipping the mutant exons can rescue the expression of dystrophin and serves as a treatment for the disease.

A number of molecular approaches have been developed to change the genetic codes on the chromosome DNA. One of such approaches utilizes the so-called “base editors”, which are engineered by fusing a nucleotide deaminase (such as cytidine deaminase or adenosine deaminase) with a Cas9 nickase. The most widely and best-characterized Cas9 is the one from Streptococcus pyogenes (SpCas9), which requires an NGG protospacer adjacent motif (PAM) sequence on the target DNA, thereby restricting the targetable genomic loci.

There exists a need to improve base editors, including their Cas9 variants, the deaminase specificity and efficiency, and the infrastructure for AAV packaging and delivery. A more effective combination of Cas9 variant, deaminase domain and the split version for AAV packaging would be helpful in correcting gene mutations and serving as a treatment for genetic diseases.

The Technology

Dr. Renzhi Han and colleagues have developed an improved adenine base editor (iABE-NG) that uses an SpCas9 variant to correct mutations in genetic diseases. The iABE-NG includes an improved SpCas9-NG which carries all the mutations in SpCas9-NG and R1335Q, a single adenine deaminase with A56G and V82G mutations which eliminates the off-target RNA editing activities, and a super-fast Gp41-1 intein split for AAV packaging. The iABE-NG allows targeted in vivo editing of a single nucleotide from A to G or T to C with high efficiencies at NG PAM sites. The team demonstrated that the recombinant AAV carrying iABE-NG can be used to correct dystrophin point mutations in a mouse model of DMD and cultured human cells. Significantly, the team demonstrated that a single injection of the recombinant AAV particles at 5 weeks of age converted over 95% of cardiomyocytes from dystrophin-negative to dystrophin-positive at 10 months of age.

Beyond treating DMD, the novel recombinant AAV with iABE-NG also holds the promise to correct gene mutations in treatments of various diseases, such as Cystic fibrosis, Phenylketonuria (PKU), Sickle-cell anemia, Huntington’s disease, Myotonic dystrophy, Hemophilia A, X-linked dilated cardiomyopathy, and familial dilated cardiomyopathy.

Patent Status

A PCT patent application is pending.

Commercial Applications

• Treatment of genetic diseases

Benefits/Advantages

• Can correct genetic mutations
• AAV delivery provides low immunogenicity and minimal integration risk
• Has gene therapy applications

Publications

• Nature Communications 2021 May;12:3719 (https://www.nature.com/articles/s41467-021-23996-y)
• Mol Ther. 2019 Aug 7; 27:(8)1407-1414 (https://doi.org/10.1016/j.ymthe.2019.05.001)