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

Amr A. Abdeen,Micah J. Dombroe,David Fiflis,Tugce Akcan,Madelyn Goedland,Amritava Das,Tausif Alam,Jared Carlson-Stevermer,Benjamin I Grindel,Heidi Kletzien,Lucille Kohlenberg,Shivani Saxena,Krishanu Saha

doi:10.1038/s41467-020-20065-8

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.

Highlights

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles
We focus on infantile-onset Pompe disease, an autosomal recessive glycogen storage disorder caused by multiple mutations in the acid-α-glucosidase (GAA) gene
To correct two endogenous alleles within the same cell, several clonal isogenic induced pluripotent stem cells (iPSCs) lines were generated by CRISPR–Cas[9] gene editing of an iPSC line derived from a patient with infantile-onset Pompe disease[47]

Summary

Introduction

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. Gene therapies typically involve the editing of a single allele[1], or delivery of exogenous genetic material (through nucleic acid delivery, viruses, or ex vivo engineered cells) to overexpress the gene of interest or suppress translation of the defective allele[1] The development of these strategies traditionally starts with studies in animal models, such studies are frequently insufficient for genetic disorders that are polygenic— involving different mutations in different alleles that exacerbate a diseased phenotype. Such studies have produced robust methods to understand the genomic changes, downstream gene expression, and phenotypic changes from editing a single allele, as has been demonstrated in several prior studies[18,19,20,21,22,23] Some of these studies, reveal that unintended genomic deletions and translocations are potential outcomes at the on-target allele[24], and such unintended outcomes are predicted to be exacerbated as when making multiple cuts in the genome through the delivery of two or more different genome editors[25]. Once some of the consequences of gene correction of multiple alleles within a single cell are characterized, important questions remain regarding how to design translational studies with gene correction strategies, both for in vivo somatic gene editing strategy or for ex vivo cell therapy with autologous genecorrected cells

Methods

Results

Conclusion