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

Authors
  • Morgan, Richard A1, 2
  • Ma, Feiyang3
  • Unti, Mildred J4
  • Brown, Devin4
  • Ayoub, Paul George4
  • Tam, Curtis4
  • Lathrop, Lindsay4
  • Aleshe, Bamidele4
  • Kurita, Ryo5
  • Nakamura, Yukio5
  • Senadheera, Shantha4
  • Wong, Ryan L2
  • Hollis, Roger P4
  • Pellegrini, Matteo3
  • Kohn, Donald B2, 4, 6, 7
  • 1 Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA.
  • 2 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
  • 3 Molecular Biology Institute Interdepartmental Doctoral Program, University of California, Los Angeles, Los Angeles, CA 90095, USA.
  • 4 Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
  • 5 Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan. , (Japan)
  • 6 Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
  • 7 The Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, USA.
Type
Published Article
Journal
Molecular Therapy — Methods & Clinical Development
Publisher
Elsevier
Publication Date
Jun 12, 2020
Volume
17
Pages
999–1013
Identifiers
DOI: 10.1016/j.omtm.2020.04.006
PMID: 32426415
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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. © 2020 The Author(s).

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