Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences

Richard A. Morgan; Feiyang Ma; Mildred J. Unti; Devin Brown; Paul George Ayoub; Curtis Tam; Lindsay Lathrop; Bamidele Aleshe; Ryo Kurita; Yukio Nakamura; Shantha Senadheera; Ryan L. Wong; Roger P. Hollis; Matteo Pellegrini; Donald B. Kohn

Molecular Therapy: Methods & Clinical Development (Jun 2020)

Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences

Richard A. Morgan,
Feiyang Ma,
Mildred J. Unti,
Devin Brown,
Paul George Ayoub,
Curtis Tam,
Lindsay Lathrop,
Bamidele Aleshe,
Ryo Kurita,
Yukio Nakamura,
Shantha Senadheera,
Ryan L. Wong,
Roger P. Hollis,
Matteo Pellegrini,
Donald B. Kohn

Affiliations

Richard A. Morgan: Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Corresponding author: Richard A. Morgan, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA.
Feiyang Ma: Molecular Biology Institute Interdepartmental Doctoral Program, University of California, Los Angeles, Los Angeles, CA 90095, USA
Mildred J. Unti: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Devin Brown: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Paul George Ayoub: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Curtis Tam: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Lindsay Lathrop: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Bamidele Aleshe: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Ryo Kurita: Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
Yukio Nakamura: Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
Shantha Senadheera: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Ryan L. Wong: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Roger P. Hollis: Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Matteo Pellegrini: Molecular Biology Institute Interdepartmental Doctoral Program, University of California, Los Angeles, Los Angeles, CA 90095, USA
Donald B. Kohn: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; The Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, USA; Corresponding author: Donald B. Kohn, MD, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Journal volume & issue: Vol. 17
pp. 999 – 1013

Abstract

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Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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