The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies

Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34+ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8+ and CD4+ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8+ and CD4+ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34+ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma

Effect of Insulators on the Expression of betaAS3 in Lentiviral Gene Therapy for Sickle Cell Disease

Insulators can be used to increase the therapeutic potential of gene therapy for sickle cell disease (SCD) by improving safety and resisting transgene silencing. Four experimental lentiviral vectors (LVs) carrying the betaAS3 transgene were engineered to contain the FB and Ank small insulator elements. When evaluated in single vector copy MEL cell clones, the Ank R LV demonstrated barrier activity that most closely resembled that observed with the positive control 1.2kb cHS4 insulator. Erythrocytes derived from human SCD hematopoietic stem/progenitor cells (HSPCs) transduced with the Ank R LV did not yield higher betaAS3 expression or phenotypic correction of the sickle shape than levels produced without a barrier insulator. Since the benefits of the Ank insulator in the betaAS3 LV are yet to be realized in primary cells, future experiments using serial transplants of murine HSPCs are needed to determine the extent of the barrier activity in the Ank R LV

Effect of Insulators on the Expression of betaAS3 in Lentiviral Gene Therapy for Sickle Cell Disease

Insulators can be used to increase the therapeutic potential of gene therapy for sickle cell disease (SCD) by improving safety and resisting transgene silencing. Four experimental lentiviral vectors (LVs) carrying the betaAS3 transgene were engineered to contain the FB and Ank small insulator elements. When evaluated in single vector copy MEL cell clones, the Ank R LV demonstrated barrier activity that most closely resembled that observed with the positive control 1.2kb cHS4 insulator. Erythrocytes derived from human SCD hematopoietic stem/progenitor cells (HSPCs) transduced with the Ank R LV did not yield higher betaAS3 expression or phenotypic correction of the sickle shape than levels produced without a barrier insulator. Since the benefits of the Ank insulator in the betaAS3 LV are yet to be realized in primary cells, future experiments using serial transplants of murine HSPCs are needed to determine the extent of the barrier activity in the Ank R LV

Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells

Sickle cell disease (SCD) can be cured by allogeneic hematopoietic stem cell transplant. However, this is only possible when a matched donor is available, making the development of gene therapy using autologous hematopoietic stem cells a highly desirable alternative. We used a culture model of human erythropoiesis to directly compare two insulated, self-inactivating, and erythroid-specific lentiviral vectors, encoding for γ-globin (V5m3-400) or a modified β-globin (βAS3-FB) for production of antisickling hemoglobin (Hb) and correction of red cell deformability after deoxygenation. Bone marrow CD34+ cells from three SCD patients were transduced using V5m3-400 or βAS3-FB and compared with mock-transduced SCD or healthy donor CD34+ cells. Lentiviral transduction did not impair cell growth or differentiation, as gauged by proliferation and acquisition of erythroid markers. Vector copy number averaged approximately one copy per cell, and corrective globin mRNA levels were increased more than sevenfold over mock-transduced controls. Erythroblasts derived from healthy donor and mock-transduced SCD cells produced a low level of fetal Hb that was increased to 23.6 ± 4.1% per vector copy for cells transduced with V5m3-400. Equivalent levels of modified normal adult Hb of 17.6 ± 3.8% per vector copy were detected for SCD cells transduced with βAS3-FB. These levels of antisickling Hb production were sufficient to reduce sickling of terminal-stage red blood cells upon deoxygenation. We concluded that the achieved levels of fetal Hb and modified normal adult Hb would likely prove therapeutic to SCD patients who lack matched donors

The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies

The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies

Modification of hematopoietic stem/progenitor cells with CD19-specific chimeric antigen receptors as a novel approach for cancer immunotherapy.

Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1-2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity

Modification of Hematopoietic Stem/Progenitor Cells with CD19-Specific Chimeric Antigen Receptors as a Novel Approach for Cancer Immunotherapy

Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1–2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity