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Updated: Nov 26, 2025
Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development
Published on: March 5, 2017
Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases
Jared Carlson-Stevermer1,2, Amritava Das1,3, Amr A Abdeen1
1Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA.
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.
Insights
Precise gene editing can correct multiple gene variants in single cells, offering a potential therapy for complex genetic disorders. Careful design is crucial for effective in vivo delivery and minimizing adverse events.
Area of Science:
- Genetics
- Molecular Biology
- Bioengineering
Background:
- Compound heterozygous recessive and polygenic diseases involve multiple gene variants.
- Current genome editing strategies may lead to mixed genotypes and adverse events when targeting multiple alleles.
- Developing precise methods for correcting multiple gene variants is essential for treating complex genetic diseases.
Purpose of the Study:
- To demonstrate the precise correction of two distinct mutant alleles within a single human cell using Cas9-ribonucleoprotein (RNP)-based genome editors.
- To evaluate the therapeutic potential of gene-corrected cells in an induced pluripotent stem cell (iPSC) model of Pompe disease.
- To identify key parameters for effective in vivo delivery of genome editors for treating genetic diseases.
Main Methods:
- Utilized Cas9-ribonucleoprotein complexes for precise genome editing.
- Established an induced pluripotent stem cell model for Pompe disease.
- Developed a quantitative in silico model to simulate in vivo delivery into the developing human infant liver.
Main Results:
- Achieved precise correction of two distinct mutant alleles in a single human cell.
- Corrected cells in the Pompe disease model expressed the corrected transcript from both alleles, enabling enzymatic cross-correction.
- In silico modeling identified progenitor targeting, delivery efficiency, and suppression of imprecise editing as critical parameters for therapeutic efficacy.
Conclusions:
- Precise gene editing of multiple distinct gene variants within a single cell is feasible.
- Appropriate design of genome editing strategies, considering in vivo delivery parameters, can lead to highly efficacious therapies for complex genetic diseases.
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