Quantifying the mutational landscape of retroviral and lentiviral vectors in gene therapy patients

Kevyn L Hart; Ralph Valentine Crisostomo; Annika Mittelhauser; Lingyu Zhan; Nika Kononov; Kathryn Bradford; Donald B Kohn

doi:10.1016/j.ymthe.2025.05.034

Quantifying the mutational landscape of retroviral and lentiviral vectors in gene therapy patients

Mol Ther. 2025 Sep 3;33(9):4459-4470. doi: 10.1016/j.ymthe.2025.05.034. Epub 2025 May 30.

Authors

Kevyn L Hart¹, Ralph Valentine Crisostomo², Annika Mittelhauser³, Lingyu Zhan⁴, Nika Kononov⁵, Kathryn Bradford⁶, Donald B Kohn⁷

Affiliations

¹ Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Molecular Biology Interdepartmental Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁴ Department of Psychiatry, Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA 90095, USA; The Collaboratory, Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ The Collaboratory, Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at 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 90095, USA. Electronic address: e-dkohn1@mednet.ucla.edu.

PMID: 40450520
PMCID: PMC12432904 (available on 2026-09-03)
DOI: 10.1016/j.ymthe.2025.05.034

Abstract

Adenosine deaminase severe combined immunodeficiency (ADA-SCID) is a monogenic disorder caused by mutations in the ADA gene. Gene therapy using γ-retroviral and lentiviral vector gene addition approaches have shown curative results. We sequenced the ADA transgene in transduced CD3⁺ T cells, and in peripheral blood cells from patients treated with autologous CD34⁺ cells transduced with either a γ-retroviral or lentiviral ADA gene vector to assess transgene mutational profiles. In both CD3⁺ T cells and ADA-SCID patients' cells treated with the lentiviral vector, we observed significantly higher occurrences of guanine (G)-to-adenosine (A) base substitutions than with the γ-retroviral vector. We hypothesized that this G-to-A mutational signature was due to the APOBEC3 cytosine deaminase protein family. By knocking out APOBEC3 genes in HEK239T packaging cells, APOBEC3-mediated mutagenesis decreased by 91.2% along the transgene in CD34⁺ transduced cells in comparison to CD34⁺ cells transduced with lentiviral supernatant packaged in parental HEK293T cells.

Keywords: ADA-SCID; APOBEC3; gene therapy; lentiviral vectors; reverse transcriptase.

MeSH terms

APOBEC Deaminases
Adenosine Deaminase* / deficiency
Adenosine Deaminase* / genetics
Agammaglobulinemia* / genetics
Agammaglobulinemia* / therapy
Genetic Therapy* / methods
Genetic Vectors* / genetics
HEK293 Cells
Humans
Lentivirus* / genetics
Mutation*
Retroviridae* / genetics
Severe Combined Immunodeficiency* / genetics
Severe Combined Immunodeficiency* / therapy
T-Lymphocytes / metabolism
Transduction, Genetic
Transgenes

Substances

Adenosine Deaminase
APOBEC3 proteins, human
APOBEC Deaminases

Supplementary concepts

Severe combined immunodeficiency due to adenosine deaminase deficiency