CRISPR Genome Engineering for Retinal Diseases

CRISPR Genome Engineering for Retinal Diseases

September 22, 2022

Novel gene therapy treatments for inherited retinal diseases have been at the forefront of translational medicine over the past couple of decades.

Since the discovery of CRISPR mechanisms and their potential application for the treatment of inherited human conditions, it seemed inevitable that advances would soon be made using retinal models of disease.

The development of CRISPR technology for gene therapy and its increasing potential to selectively target disease-causing nucleotide changes has been rapid.

A series of recent discoveries harnessing the adaptive immune system of prokaryotes to perform targeted genome editing is having a transformative influence across the biological sciences.

The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy.

Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity.

While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy.

Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation.

The latest research in the area of clustered regularly interspaced short palindromic repeats (CRISPR) technology may provide a paradigm to accelerate the development of CRISPR–associated protein 9 (CRISPR-Cas9) therapies to correct patient-derived induced pluripotent stem cells in inherited retinal dystrophies, according to Erin Burnight, PhD, associate research scientist in the Department of Ophthalmology and Visual Sciences at the University of Iowa Carver College of Medicine in Iowa City.

Although the ability to select for corrected disease-causing variants is effective in establishing disease models and developing cell therapies for inherited retinal diseases, there is a shortcoming, Burnight said: In some cases, placement of the selection cassette can interfere with the expression from the targeted locus.

In light of this, she and her colleagues theorized that they could address this interference at the intended locus using a cotargeting strategy that would delivering CRISPR-Cas9 reagents to 2 locations.

One targeted the locus of interest; the second, a separate locus. “Thus, this approach produced a cotargeting strategy using the adeno-associated virus vector [AAVS1] safe harbor site as the selection locus,” she said.

How they did it

The investigators reported that they made a plasmid carrying a Streptococcus pyogenes Cas9 expression cassette and single-guide RNA expression cassettes that targeted the locus of interest and the AAVS1 site.

Five sites in total were targeted: CRX, GRK1, VSX2, USH2A, and CEP290. The CRISPR-Cas9 expression plasmid, a donor template carrying homologous sequence to the locus of interest, and a donor template carrying a puromycin selection cassette flanked by AAVS1 homology sequence were delivered to human cells via cationic lipid transfection and cultured under puromycin selection for 10 to 14 days.

Genomic DNA was isolated from puromycin-resistant clones and screened for modification at the locus of interest via polymerase chain reaction.

The investigators successfully cotargeted the 5 sites, and the efficiency of the cotargeting effort varied by locus, Burnight said.

“We cloned and screened single-guide RNAs targeting at the 5 loci. We achieved successful cotargeting at all 5 loci as revealed by amplification of donor-target junctions and RFLP [restriction fragment length polymorphism] analysis,” she said.

“The clonal analysis at CRX, GRK1, and VSX2 showed cotargeting in 5%, 20%, and 35% of puromycin-resistant clones, respectively.” The investigators concluded that their results may provide a paradigm to develop CRISPR-Cas9 treatments for inherited retinal dystrophies.