Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I

Natalia Gomez-Ospina,Nathalie Mostrel,Kazuki Sawamoto,Gang Bao,Channabasavaiah B Gurumurthy,Ciaran Lee,Carlos J Suarez,Laure Aurelian,Nitin Raj,Laura D Attardi,Sruthi Mantri,Shaukat Khan,Samantha G Scharenberg,Rolen M Quadros,Shunji Tomatsu,Rasmus O Bak,Matthew H Porteus

doi:10.1038/s41467-019-11962-8

Abstract

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient’s own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.

Highlights

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments
RNP complexes consisting of 2′-Omethyl 3′phosphorothioate-modified CCR5 sgRNA21 and Cas[9] protein were electroporated into cord blood-derived (CB) and adult peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) (PB)
The efficiency of double-strand DNA break (DSB) generation by our CCR5 RNP complex was estimated by measuring the frequency of insertions/ deletions (Indel) at the predicted cut site

Summary

Introduction

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. The peroxisomal disorder X-linked adrenoleukodystrophy has been successfully treated by lentiviral transduced autologous HSPCs though supra-normal expression of the missing enzyme is probably not critical as cross-correction is not a feature of this disease[10,11] This autologous approach eliminates the need to find immunologically matched donors and reduces some of the potential complications from allogeneic transplants. When targeted to the sequence determined by the sgRNA, Cas[9] creates a double-stranded DNA break, thereby stimulating homologous recombination with a designed donor DNA template that contains the desired genetic modification embedded between homology arms centered at the break site This process, termed “homologous recombination-mediated genome editing” (HR-GE) is most often used for in situ gene correction and has been hailed as a tool to treat monogenic diseases. Its disruption has no effect on cell proliferation and no known potential for oncogenic transformation

Methods

Results

Conclusion