FDA-approved gene therapy trials have treated autosomal-recessive (i.e., loss-of-function) disorders by supplementation with the wild-type (WT) version of the mutant gene. For patients with autosomal-dominant (ad) gain-of-function disorders, the best hope for a cure is genome surgery that repairs or removes the malfunctioning genes at the root of the disease. Currently, that hope lies in CRISPR/Cas9-based gene editing {DiCarlo, Mahajan & Tsang, J Clin Invest. 2018;128:2177}. The strength of the first-generation CRISPR-based therapy (CRISPR1.0; Fig. 1)?its mutation-specificity?is also its greatest weakness. This is because the therapeutic components for each mutation (both the guide RNA (gRNA) and the repair template) need to be custom-designed, engineered, tested, and FDA-approved. This presents a considerable and costly challenge for the many ad diseases caused by a slew of different mutations. For example, the blinding Best vitelliform macular dystrophy (VMD) disorder is caused by any 1 of 250 different mutations in the rhodopsin (BEST1) gene. Treatment of all patients would, therefore, require that 250 sets of CRISPR1.0 components be engineered, validated, and FDA- approved. To overcome this major limitation, we developed CRISPR2.0 (Fig.2), a mutation nonspecific strategy. Unfortunately, CRISPR2.0 is not allele-specific and so eliminates both the mutant and WT alleles. As a result, CRISPR2.0 requires gene supplementation, which leads to variable expression of the rescued gene and sustainability concerns. We now propose to develop a third-generation CRISPR-based strategy, CRISPR3.0 (Fig. 2), that, like CRISPR2.0, is mutation nonspecific. However, CRISPR3.0 is allele-specific and therefore ablates the mutant, disease-causing cis allele while leaving the WT allele intact to support normal function. We hypothesize that CRISPR3.0 chromosome-specific genome surgery will produce a more sustained therapeutic response compared to the CRISPR2.0 supplementation strategy.
Autosomal dominant conditions are debilitating, and there is no treatment available. CRISPR gene editing as it currently stands is unrealistic to translate to a clinical setting due to the gene- and mutation-specific guide RNA (gRNA) targeting requirements and delivery. Our strategy will address this problem by providing an efficacious way to treat autosomal dominant conditions; identifying and optimizing the ideal CRISPR delivery best suited for these conditions; and enabling treatment in a manner that is not mutation specific.
