β-globin gene transfer to human bone marrow for sickle cell disease
Zulema Romero, … , Thomas D. Coates, Donald B. Kohn
Zulema Romero, … , Thomas D. Coates, Donald B. Kohn
Published July 1, 2013
Citation Information: J Clin Invest. 2013;123(8):3317-3330. https://doi.org/10.1172/JCI67930.
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Research Article Hematology

β-globin gene transfer to human bone marrow for sickle cell disease

Abstract

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin–like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2–3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.

Authors

Zulema Romero, Fabrizia Urbinati, Sabine Geiger, Aaron R. Cooper, Jennifer Wherley, Michael L. Kaufman, Roger P. Hollis, Rafael Ruiz de Assin, Shantha Senadheera, Arineh Sahagian, Xiangyang Jin, Alyse Gellis, Xiaoyan Wang, David Gjertson, Satiro DeOliveira, Pamela Kempert, Sally Shupien, Hisham Abdel-Azim, Mark C. Walters, Herbert J. Meiselman, Rosalinda B. Wenby, Theresa Gruber, Victor Marder, Thomas D. Coates, Donald B. Kohn

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Figure 6

In vivo assessment of CCL-βAS3-FB LV transduction of BM CD34+ cells.

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In vivo assessment of CCL-βAS3-FB LV transduction of BM CD34+ cells. (...
(A) Engraftment of human cells in NSG mice. BM cells isolated from mice from each transplant group (nos. 1–6) were analyzed by flow cytometry to measure the percentage of human CD45+ cells among all CD45+ cells in the marrow (human and murine) as a measurement of engraftment. Mock transduced, white triangles; CCL-βAS3-FB transduced, black triangles. BM samples from HD were used in transplants 3, 4, and 6 and from SCD donors in transplants 1, 2, and 5. (B) Immunophenotypic analysis of human cells isolated from NSG mice transplanted with transduced BM CD34+ cells. Flow cytometry was used to enumerate the percentage of the human CD45+ cells that were positive for the markers of B-lymphoid cells (CD19, white), myeloid progenitors (CD33, light gray), hematopoietic progenitors (CD34, dark gray), and erythroid cells (CD71, black). Mean ± SD are shown of 3 independent experiments. Mock, n = 4; βAS3-FB, n = 8 mice. (C) VC/cell in human cells cultured from NSG mice transplanted with transduced BM CD34+ cells. Black circles represent samples from mice transplanted with HD BM, and white squares represent mice transplanted with SCD BM. All the human cells examined from mock-transduced mice were negative for VC analysis by qPCR. (D) HBBAS3 mRNA expression measured by qRT-PCR from cells transduced to different VC/cell. Five independent transductions are shown. HD, black circles (n = 6); SCD, white squares (n = 4).