Preclinical safety assessment of modified gamma globin lentiviral vector-mediated autologous hematopoietic stem cell gene therapy for hemoglobinopathies
Mohammad Shadid, Archana Shrestha, Punam Malik
Abstract
Previously, we reported the development of a human Aγ-globin gene lentivirus (LV), GbG, which expresses high levels of HbF to correct the sickle cell anemia (SCA) phenotype in the Berkeley SCA mouse model, and then modified the γ-globin gene by substituting glycine at codon 16 with aspartic acid in the Aγ-globin gene to generate GbGM LV. In the present study, we evaluated the long-term safety of human Aγ-globin gene carrying GbGM LV in wild-type mice after primary and secondary transplants of GbGM-modified hematopoietic stem cells (HSC) over 18 months.
Introduction
Sickle cell disease (SCD) is an autosomal recessive genetic disorder caused by a mutation in the β-globin chain genes leading to sickle hemoglobin production (hemoglobin S or HbS) instead of the normal adult hemoglobin, HbA. The single nucleotide mutation in the 6th codon of the β-globin gene results in the substitution of glutamic acid to valine, thus forming the defective HbS. HbS polymerizes upon deoxygenation, changing the shape of the round RBC to sickle shapes and causing deformation of red blood cells (RBC), microvascular occlusions, ischemia, and multi-organ damage [1].
Materials and method
Test and control articles
The test article is a mutant Aγ-globin gene carrying LV GbGM that expresses human γ-globin with a point mutation at exon 1. This point mutation was made as in our previously published GbG LV [18]. The RV encodes green fluorescent protein (GFP), and the plasmid pRSF91GFP.pre* was kindly provided by Dr. Christopher Baum and used by the CCHMC’s Vector Production Facility to produce the pRSF9 l GFP.pre* vector pseudotyped with the ecotropic envelope (pRSF91GFP.pre*/Eco). This vector is a spleen focus forming virus (SFFV) long terminal repeat driven RV that was used as a positive control in the insertional mutagenesis study described by Modlich et al. [20].
Results
Efficient transduction and engraftment in primary recipient mice
The VCN for the GbGM and SFFV RV in the infused LSK cells were 3.5 and 4.4, respectively (Fig 1A). As expected, no vector DNA was detected in the Mock group samples. After transplantation, the engraftment was evaluated at week 7 and week 17 in peripheral blood (PB) leukocytes (S1 Table). PB engraftment (CD45.2+ donor leukocytes) in the recipient mice for all the groups was ~91% at week 7 and increased to 96% by week 17. The primary recipient mice were followed for 8 months, after which animals were sacrificed and a portion of harvested BM cells were used for VCN and engraftment analysis while the rest were used for secondary transplant.
Discussion
Autologous HSPC transduced ex vivo with an LV represents a promising approach to treat monogenic disorders such as SCA [21]. However, one needs to rigorously rule out the risk of hematological malignancy, which is dependent on the vector design. The risk is expected to be negligible with an LV that carries erythroid-specific promoter/enhancer elements. Herein, we demonstrated the preclinical safety of a modified γ-globin LV, GbGM, in mice that underwent primary and secondary transplants.
Acknowledgments
We thank the staff in the Translational Core Laboratory, specifically, Drs. Christoph Burkart and Elke Grassman, Diana Nordling for performing this safety study, and Dr. Paritha Arumugam for providing the Translational Core Laboratory expert advice on LV gene transfer and analysis.
Citation: Shadid M, Shrestha A, Malik P (2024) Preclinical safety assessment of modified gamma globin lentiviral vector-mediated autologous hematopoietic stem cell gene therapy for hemoglobinopathies. PLoS ONE 19(7): e0306719. https://doi.org/10.1371/journal.pone.0306719
Editor: Chen Ling, Fudan University, CHINA
Received: April 24, 2024; Accepted: June 5, 2024; Published: July 8, 2024
Copyright: © 2024 Shadid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data that supports the findings of this work is available in the supplementary material of this article. Additional data related to this study can be available upon request to the authors.
Funding: The author(s) received no specific funding for this work.
Competing interests: MS and AS are employees of Aruvant Sciences. PM has served as a consultant Aruvant Sciences.
