Behavioral and molecular effects of Ubtf knockout and knockdown in mice.

The UBTF E210K neuroregression syndrome is caused by de novo dominant mutations in UBTF (NM_014233.3:c.628G > A, p.

Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells.

Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.

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.

High-efficiency transduction of rhesus hematopoietic repopulating cells by a modified HIV1-based lentiviral vector.

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with χHIV vectors.

Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.

β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α₂β₂;HbA). Increased levels of fetal hemoglobin (α₂γ₂;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34(+) cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major.

A zinc-finger transcriptional activator designed to interact with the gamma-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts.

Fetal hemoglobin (HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. We used an in vitro culture model of human erythropoiesis in which late-stage erythroblasts are derived directly from human CD34(+) hematopoietic cells to evaluate HbF production. This system recapitulates expression of globin genes according to the developmental stage of the originating cell source.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter.

Correction of murine sickle cell disease using gamma-globin lentiviral vectors to mediate high-level expression of fetal hemoglobin.

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model.

Globin lentiviral vector insertions can perturb the expression of endogenous genes in beta-thalassemic hematopoietic cells.

Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as beta-thalassemia and sickle cell disease may hold a similar risk.

In vivo bioluminescence imaging for early detection and monitoring of disease progression in a murine model of neuroblastoma.

Background: We evaluated the potential of bioluminescence imaging (BLI) for early tumor detection, demonstrating occult sites of disseminated disease and assessing disease progression in a murine model of neuroblastoma.

Methods: Neuroblastoma cells engineered to express the enzyme firefly luciferase were used to establish localized tumors and disseminated disease in SCID mice. Bioluminescent signal intensity was measured at serial time points, and compared with traditional methods of evaluating tumor growth.

Extended beta-globin locus control region elements promote consistent therapeutic expression of a gamma-globin lentiviral vector in murine beta-thalassemia.

Since increased fetal hemoglobin diminishes the severity of beta-thalassemia and sickle cell anemia, a strategy using autologous, stem cell-targeted gene transfer of a gamma-globin gene may be therapeutically useful. We previously found that a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region (LCR) totaling 1.7 kb could correct murine beta-thalassemia.

Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells.

Successful gene therapy of beta-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of beta-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer.

The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number.

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines.