Although gene editing has enormous clinical promise, it still faces many obstacles that must be overcome before broad translation. For example, the genome editing field is continuously working to increase the safety of the technique. One way to do that is to control the duration of gene editing activity. However, achieving precise temporal control over these platforms is challenging.
Scientists currently lack tools that can precisely control the duration of gene editing therapies, to halt their effects after a few months or years. Researchers have developed several potential solutions, including degradable protein- or RNA-based systems, but existing approaches face limitations such as only being applicable in the liver.
Now, a new gene editing system, named PRINCE, with inducible nuclease proteins and guide RNAs, enables researchers to control the duration and specificity of gene therapies precisely with small molecules—potentially addressing a longstanding safety concern in the field.
This work is published in Science Translational Medicine in the paper, “Coordinated regulation using small-molecule drugs enables controlled therapeutic genome editing and enhanced genomic precision in situ.“
In the PRINCE CRISPR-Cas gene editing system, expression of the nuclease and the guide RNA is separately inducible by two approved small molecule drugs, enabling both temporal control and reduced off-target editing.
The platform was stable for as long as two years after genomic integration in cultured human cells. In addition, a smaller system, called Little Prince, showed promising signs of efficacy in humanized mouse models of elevated cholesterol levels and age-related macular degeneration. Little Prince uses compact nucleases that can be delivered in a single adeno-associated viral vector (AAV) for delivery.
In two mouse models, Little Prince reduced excessive cholesterol levels in mice with genetic hypercholesterolemia and showed signs of reducing lesion size in rodents with laser-induced choroidal neovascularization—a model of age-related macular degeneration.
More specifically, Little Prince “ameliorated pathological phenotypes of hypercholesterolemia (average reductions of 45% and 47% in serum total cholesterol and low-density lipoprotein cholesterol, respectively) and neovascular age-related macular degeneration, with significantly reduced lesion size and leakage (P < 0.0001).”
The system produced fewer off-target edits and lower off-target editing frequencies than constitutive nuclease expression, highlighting the utility of precise temporal control.
“PRINCE and Little Prince also provide […] capabilities that might also be useful for research objectives that include lineage tracing and conditional genetic engineering work,” the authors noted. These results, they asserted, position PRINCE and Little Prince as controlled genome editing platforms with potential for in vivo, particularly in situ, therapeutic applications.
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