Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases

Abstract

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

Utility of iPSC-Derived Cells for Disease Modeling, Drug Development, and Cell Therapy

“…However, there are challenges associated with miniaturization including the survival of cells plated in low numbers, evaporation of culture media due to low volume of reagents and accuracy in dispensing low volumes of compounds. The use of this technology in drug discovery has allowed the identification of many new compounds for the potential treatment of various diseases including Zika virus [ 66 ], SMA [ 51 ], as well as understanding cellular processes and developmental stages using genome editing strategies [ 72 ].…”

“…The generation of isogenic controls, whereby correcting gene mutations back to wild-type, provide the best controls for disease models. Such disease models could also be used in combination with in silico approaches for high-throughput drug discovery and drug development studies [ 72 ].…”

Synthetic Biology Approaches for Engineering Next-Generation Adenoviral Gene Therapies

“…26 Ad delivery of multiplexed CRISPR-Cas systems may allow genetic treatment of polygenic conditions. 47,50 Synthetic biology may help construct superior Ad vectors and enable polygenic Ad therapies.…”

“…As has already been seen in the field of AAV gene therapy, high-throughput experimental techniques could provide training data for machine learning models which might extract superior Ad vector designs . Ad delivery of multiplexed CRISPR-Cas systems may allow genetic treatment of polygenic conditions. , Synthetic biology may help construct superior Ad vectors and enable polygenic Ad therapies.…”

“…In particular, de novo protein design software and AI-guided sequence engineering could gain prominence. Multiplexed CRISPR-Cas parts in gutless Ad chassis could give Ad synthetic biology a strong foothold as a player in the emerging field of polygenic gene therapy . These possibilities in Ad synthetic biology represent an opportunity for the field to grow and to potentially transform the future landscape of gene therapy.…”