Biomedical subjects

Derek A Persons

Publications and source records attributed to Derek A Persons.

17 recordsLinked to original sources

PubMed2005

Role of erythropoietin receptor signaling in Friend virus-induced erythroblastosis and polycythemia.

Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed. Subsequently, we showed that a truncated form of the stem-cell kinase receptor (sf-STK) is required for susceptibility to Friend disease. Given the requirement for sf-STK, we sought to establish the in vivo significance of gp55-mediated activation of the EPOR. We found that the cytoplasmic tyrosine residues of the EPOR, and signal transducer and activator of transcription-5 (STAT5), which acts through these sites, are not required for Friend virus-induced erythroblastosis. The EPOR itself was required for the development of erythroblastosis but not for gp55-mediated erythroid proliferation. Interestingly, the murine EPOR, which is required for gp55-mediated Ba/F3-cell proliferation, was dispensable for erythroblastosis in vivo. Finally, gp55-mediated activation of the EPOR and STAT5 are required for Friend virus-induced polycythemia. These results suggest that Friend virus activates both sf-STK and the EPOR to cause deregulated erythroid proliferation and differentiation.

Animals↗

PubMed2005

Transgenic mice with pancellular enhanced green fluorescent protein expression in primitive hematopoietic cells and all blood cell progeny.

Transgenic mice homogeneously expressing enhanced green fluorescence protein (EGFP) in primitive hematopoietic cells and all blood cell progeny, including erythrocytes and platelets, have not been reported. Given previous data indicating H2Kb promoter activity in murine hematopoietic stem cells (HSCs), bone marrow (BM), and lymphocytes, an H2Kb enhancer/promoter EGFP construct was used to generate transgenic mice. These mice demonstrated pancellular EGFP expression in both primitive BM Sca-1+Lin-Kit+ cells and side population (SP) cells. Additionally, all peripheral blood leukocytes subsets, erythrocytes, and platelets uniformly expressed EGFP strongly. Competitive BM transplantation assays established that transgenic H2Kb-EGFP HSCs had activity equivalent to wildtype HSCs in their ability to reconstitute hematopoiesis in lethally irradiated mice. In addition, immunohistochemistry revealed EGFP transgene expression in all tissues examined. This transgenic strain should be a useful reagent for both murine hematopoiesis studies and functional studies of specific cell types from particular tissues.

Animals↗

PubMed2005

Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors.

Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3' long terminal repeat (LTR) is copied over into the 5' LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.

Animals↗

PubMed2004

Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+)cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

Animals↗

PubMed2004

Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation.

Bone and bone marrow are closely aligned physiologic compartments, suggesting that these tissues may represent a single functional unit with a common bone marrow progenitor that gives rise to both osteoblasts and hematopoietic cells. Although reports of multilineage engraftment by a single marrow-derived stem cell support this idea, more recent evidence has challenged claims of stem cell transdifferentiation and therefore the existence of a multipotent hematopoietic/osteogenic progenitor cell. Using a repopulation assay in mice, we show here that gene-marked, transplantable marrow cells from the plastic-nonadherent population can generate both functional osteoblasts/osteocytes and hematopoietic cells. Fluorescent in situ hybridization for the X and Y chromosomes and karyotype analysis of cultured osteoblasts confirmed the donor origin of these cells and excluded their generation by a fusion process. Molecular analysis demonstrated a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of seven animals studied, establishing a shared clonal origin for these ostensibly independent cell types. Our findings indicate that the bone marrow contains a primitive cell able to generate both the hematopoietic and osteocytic lineages. Its isolation and characterization may suggest novel treatments for genetic bone diseases and bone injuries.

Animals↗

PubMed2004

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. However, therapeutic consistency was compromised by chromosomal position effects on vector expression. In contrast, we show here that the majority of animals that received transplants of beta-thalassemic stem cells transduced with a new vector containing 3.2 kb of LCR sequences expressed high levels of fetal hemoglobin (17%-33%), with an average vector copy number of 1.3. This led to a mean 26 g/L (2.6 g/dL) increase in hemoglobin concentration and enhanced amelioration of other hematologic parameters. Analysis of clonal erythroid cells of secondary spleen colonies from mice that underwent transplantation demonstrated an increased resistance of the larger LCR vector to stable and variegating position effects. This trend was also observed for vector insertion sites located inside genes, where vector expression was often compromised, in contrast to intergenic sites, where higher levels of expression were observed. These data emphasize the importance of overcoming detrimental position effects for consistent therapeutic globin vector expression.

Anemia, Sickle Cell↗

PubMed2004

Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system.

High-titer, HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (< 1%), reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier, we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo, 3 rhesus macaques underwent transplantation with transduced, autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid, and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes, respectively, were observed 4 to 6 months after transplantation, consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models.

Animals↗

PubMed2004

Gene therapy for the hemoglobin disorders.

The hemoglobin disorders of beta-thalassemia and sickle cell disease together constitute the most prevalent group of human monogenic diseases. Although curative allogeneic stem cell transplantation therapy and palliative therapies have been developed for these disorders, the majority of patients still suffer significant morbidity and early mortality. The development of therapeutic approaches based on genetic manipulation of autologous stem cells therefore remains an attractive alternative. In the past 4 years, significant advances have been made toward this goal using lentiviral vectors to obtain high-level expression of complex globin gene cassettes. Therapeutic correction in murine models of both beta-thalassemia and sickle cell anemia has been achieved using this approach. These advances, coupled with progress in the ability to achieve in vivo selection of genetically modified cells, can now be evaluated in the well-developed nonhuman primate autologous transplant model. The goal in these studies is to provide preclinical safety and efficacy data prior to human clinical trials in order to maximize the likelihood of success in the context of an acceptable risk to benefit ratio. Here we review progress in each of these areas.

Animals↗

PubMed2003

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors.

One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

Animals↗

PubMed2003

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. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into beta-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel gamma-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in gamma-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with beta-thalassemia.

Animals↗

PubMed2003

Development of gene therapy for hemoglobin disorders.

The hemoglobin disorders, severe beta-thalassemia and sickle cell anemia, are prevalent monogenetic disorders which cause severe morbidity and mortality worldwide. Gene therapy approaches to these disorders envision stem cell targeted gene transfer, autologous transplantation of gene-corrected stem cells, and functional, phenotypically corrective globin gene expression in developing erythroid cells. Lentiviral vector systems potentially appear to afford adequately efficient gene transfer into stem cells and are capable, with appropriate genetic engineering, of transferring a globin gene with the regulatory elements required to achieve high-level, erythroid-specific expression. Herein are results obtained in use of lentiviral vectors to insert a gamma-globin gene into murine stem cells with phenotypic correction of the thalassemia phenotype. Further, we have developed a drug-selection system for genetically modified stem cells based on a mutant form of methylguanine, methyltransferase, which allows selective amplification of genetically modified stem cells with phenotypic correction even in the absence of myeloablation prior to stem cell transplantation. These advances provide essential preclinical data which build toward the development of effective gene therapy for the severe hemoglobin disorders.

Anemia, Sickle Cell↗

PubMed2003

Gene therapy for the hemoglobin disorders.

The hematologic disorders of beta-thalassemia and sickle cell disease are the most prevalent of human genetic diseases. Although palliative therapies and curative stem cell transplantation therapy have been developed for these disorders, treatment still remains suboptimal and many patients suffer significant morbidity and early mortality. Therefore, development of a gene therapy approach has been sought for many years. Major progress in the globin gene therapy field has been achieved by several laboratories. Using lentiviral vectors to obtain high-level expression of complex globin gene cassettes, therapeutic correction of several murine models of beta-thalassemia, and sickle cell disease was recently reported. This progress, coupled with developments in the ability to select and expand genetically modified stem cells in vivo, has advanced the possibility of gene therapy for the hemoglobin disorders in the near future. We review the developments in several areas that are critical for successful gene therapy of the hemoglobin disorders.

Anemia, Sickle Cell↗

PubMed2003

Update on gene therapy for hemoglobin disorders.

The hemoglobin disorders of beta-thalassemia and sickle cell disease together constitute the most prevalent of human monogenic diseases. Although palliative therapies and curative allogeneic stem cell transplantation therapy have been developed for these disorders, many patients still suffer significant morbidity and early mortality. Therefore, development of alternative treatment based on a gene therapy approach continues to be a worthwhile endeavor. Several laboratories have recently achieved major progress towards this goal. Using lentiviral vectors to obtain high-level expression of relatively complex globin gene cassettes, therapeutic correction of several murine models of both beta-thalassemia and sickle cell disease has been achieved. These breakthroughs, coupled with recent significant developments in the ability to select and expand genetically modified stem cells in vivo, have greatly advanced the possibility of gene therapy for the hemoglobin disorders in the near future. These advances, together with recent information regarding safety issues of retroviral gene transfer, are reviewed here.

Animals↗

PubMed2002

High-level erythroid lineage-directed gene expression using globin gene regulatory elements after lentiviral vector-mediated gene transfer into primitive human and murine hematopoietic cells.

Lentiviral vectors efficiently transduce primitive human hematopoietic cells and are capable of transferring complex genomes. Vectors were designed with hypersensitive sites containing regulatory elements from the beta-globin locus control region linked to the beta-globin gene promoter to drive expression of the enhanced green fluorescent protein marker to facilitate analysis of the pattern of gene expression in various hematopoietic lineages. Such vectors gave higher level, induced expression in mouse erythroleukemia cells than a previously described vector that utilized an enhancer from the alpha locus and the ankyrin-1 promoter [Moreau-Gaudry, F., Xia, P., Jiang, G., Perelman, N.P., Bauer, G., Ellis, J., Surinya, K.H., Mavilio, F., Shen, C.K., and Malik, P. (2001). Blood 98, 2664-2672]. The addition of gamma-globin intron sequences further augmented vector expression. Expression was also effectively targeted to the erythroid lineage in cultured human cells from peripheral blood and in mouse red blood cells in vivo, although lower levels of expression were also observed in other lineages. Thus, these newly described vectors provide a means to achieve high-level gene expression, predominantly in erythroid cells, an outcome that may have potential therapeutic application.

Animals↗

PubMed2002

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. Compared with gamma-globin, oncoretroviral vectors containing either a beta-spectrin or beta-globin promoter and the alpha-globin HS40 element, a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human gamma-globin was observed with levels of vector-encoded gamma-globin mRNA ranging from 9% to 19% of total murine alpha-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of beta-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m(alpha)(2)h(gamma)(2) molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. gamma-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a gamma-globin lentiviral vector for gene therapy of beta-thalassemia.

Animals↗

PubMed2002

Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood.

Substantial effort has been invested in developing methodologies for efficient gene transfer into human, repopulating, hematopoietic stem cells. Oncoretroviral vectors are limited by the lack of nuclear mitosis in quiescent stem cells during ex vivo transduction, whereas the preintegration complex of lentiviral vectors contains nuclear-localizing signals that permit genome integration without mitosis. We have developed a flexible and versatile system for generating lentiviral vector particles and have pseudotyped such particles with amphotropic, ecotropic, feline endogenous virus (RD114) or vesicular stomatitis virus (VSV-G) envelope proteins. Particles of all four types could be concentrated approximately 100-fold by ultracentrifugation or ultrafiltration. RD114 or amphotropic particles were more efficient than VSV-G-pseudotyped particles at transducing human cord blood CD34(+) cells and clonogenic progenitors within that population. Amphotropic particles transduced cytokine-mobilized, human peripheral blood CD34(+) cells capable of establishing hematopoiesis in immunodeficient mice more efficiently than the other two types of particles. We conclude that the use of amphotropic pseudotyped lentiviral vector particles rather than the commonly used VSV-G-pseudotyped particles should be considered in potential applications of lentiviral vectors for gene transfer into this therapeutically relevant target cell population.

Animals↗

PubMed2002

In vivo selection to improve gene therapy of hematopoietic disorders.

Successful gene therapy of hematopoietic disorders lacking intrinsic natural selection for genetically corrected cells will require efficient ex vivo gene transfer into autologous hematopoietic stem cells (HSCs). For these diseases, currently available gene transfer methodologies are unlikely to result in therapeutic numbers of corrected HSCs, especially in the setting of minimal recipient conditioning. A strategy to increase the numbers of genetically corrected HSCs in an individual is therefore highly desirable. One approach to overcome the barrier of limiting numbers of genetically corrected cells is to endow them with a competitive advantage conferred by inclusion of a 'selectable' gene in the therapeutic vector. Herein, we review recent progress in the development of in vivo selection systems, which hold promise in facilitating successful gene therapy.

Bone Marrow Diseases↗