Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

Giulia Pavani,Marine Laurent,Mario Amendola,Erika Cantelli,Peter J Lenting,Guillaume Corre,Annarita Miccio,Anna Fabiano,Magali Toueille,Jean-Paul Concordet,Aboud Sakkal

doi:10.1038/s41467-020-17552-3

Abstract

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients’ cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

Highlights

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy
To generate a DNA double-strand break (DSB) for transgene integration in the α-globin locus, we focused on Streptococcus pyogenes (Sp)Cas[9] nuclease, the only Cas in clinical trials to edit hematopoietic stem cells (HSCs) (NCT03164135; NCT03655678)
We developed an ex vivo platform for efficient gene targeting in human HSCs and robust expression of therapeutic transgenes by the erythroid lineage

Summary

Introduction

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. Genome editing technologies have a great therapeutic potential for genetic disorders, as they can fix the underlying diseasecausing mutation[3,4] This approach requires the development of countless gene-tailored editing strategies that can hinder clinical translation. The striking transcriptional activity of this locus achieved therapeutic protein levels in different preclinical models and prompted the first in vivo genome editing trial in humans (NCT03041324) Promising, this approach is hampered by: (1) presence of pre-existing antibodies against AAV capsid that precludes treatment to a significant portion of patients[8]; (2) long-term expression of synthetic nucleases in vivo, which could result in genotoxicity and trigger immune responses against transduced hepatocytes[9,10]; (3) liver conditions that can alter AAV transduction and hepatic protein expression[11,12]. From liver, autologous HSCs can be accessed for ex vivo gene manipulation and re-administration, circumventing immunological issues; a suitable locus for transgene integration (knock-in, KI) still needs to be identified

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