Engraftment Of Gene-modified Cells Research Articles

Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown for Sickle Cell Gene Therapy

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

Effects of Cyclosporine A on Lentiviral Transduction of Mouse Hematopoietic Stem Cells and Transplantation Using Nonmyeloablative Conditioning.

Achieving high level transduction of murine hematopoietic stem cells (HSCs) using lentiviral vectors has been a challenge for many laboratories. We investigated the efficiency of lentiviral transduction of murine stem cell antigen-1+ (sca-1+) cells with and without cyclosporine A (CSA), which has previously been shown to increase the transduction efficiency of other types of murine cells. Sca-1+ cells were isolated from C57BL/6 mice and transduced with lentiviral vectors encoding green fluorescent protein (GFP) at various multiplicity of infections (MOI) and with various concentrations of CSA. Twenty-four hours after a single transduction, 1.5 x 104 or 4.5 x 104 cells were plated in methylcellulose containing the appropriate cytokines for progenitor cell growth, and colonies were counted on days 8–12. In the absence of CSA only 4± 2% of progenitor colonies were GFP+. However, when CSA (10 μM) was added within 3 hours of sca-1 cell isolation, transduction efficiency increased to 44 ± 6%. Although the transduction efficiency increased 10-fold, the number of progenitor colonies significantly decreased when CSA was added (up to 90% decrease). Lower concentrations of CSA (e.g. 1 μM) were less toxic to sca-1+ cells but resulted in inconsistent transduction efficiencies. We next determined the effects of CSA when applied at various times after sca-1 cell isolation. We found that the number of sca-1+ cells decreased within the first two days of culture but then begin to increase on day 3, and by day 7 there is a 7-fold increase compared to the number of cells originally isolated. Cells cultured with virus alone had an average increase of 3.5-fold on day 7, but only 3% of cells cultured in CSA survived to day 7. Cells cultured with both virus and CSA had no viability on day 7. However, by delaying the addition of virus and CSA until day 3, a 1.4-fold increase in sca-1+ cells was observed by day 7, which was achieved without affecting the efficiency of transduction. Sca-1+ cells were then transduced with the lentiviral vector in the presence of CSA and transplanted into transgenic sickle mice using a nonmyeloablative conditioning regimen that consisted of busulfan (25 mg/kg) administered on day -1 and costimulation blockade with CTLA-4Ig and anti-CD40 ligand administered on days 0, 2, 4, and 7. We were able to achieve donor engraftment levels of 98% with a 40% engraftment of gene-modified cells. These results show that using CSA in lentiviral transductions of murine HSCs can be an effective method for increasing overall transduction efficiency, and may aid in the use of lentiviral vectors in animal studies.