Search PubMed⌕ Search

Biomedical subjects

Richard A Morgan

Publications and source records attributed to Richard A Morgan.

25 records · Page 2Linked to original sources
PubMed2002

Toxicity of a first-generation adenoviral vector in rhesus macaques.

We constructed a first-generation adenovirus vector (AVC3FIX5) that we used to assess the rhesus macaque as a nonhuman primate model for preclinical testing of hemophilia B gene therapy vectors. Although we succeeded in our primary objective of demonstrating expression of human factor IX we encountered numerous toxic side effects that proved to be dose limiting. Following intravenous administration of AVC3FIX5 at doses of 3.4 x 10(11) vector particles/kg to 3.8 x 10(12) vector particles/kg, the animals in our study developed antibodies against human factor IX, and dose-dependent elevations of enzymes specific for liver, muscle, and lung injury. In addition, these animals showed dose-dependent prolongation of clotting times as well as acute, dose-dependent decreases in platelet counts and concomitant elevation of fibrinogen and von Willebrand factor. These abnormalities may be caused by the direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in IL-6, a key regulator of the acute inflammatory response. The rhesus macaque may be a useful animal model in which to evaluate mechanisms of adenovirus toxicities that have been encountered during clinical gene therapy trials.

Adenoviruses, Human↗
PubMed2002

Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference.

Mosquitoes transmit numerous viral pathogens to humans including dengue virus which affects approximately 50 million individuals per year. Inhibition of viral gene expression within an insect host could be used to block virus replication and subsequent transmission of the pathogen to humans. A naturally occurring gene silencing mechanism triggered by double-stranded RNA (dsRNA), RNA interference (RNAi), has recently been described in a number of species including Drosophila. To ascertain if dsRNA-triggered RNAi is present in mosquito cells, we used Aedes albopictus C6/36 cells, and to investigate the feasibility of blocking viral gene expression and replication, we used two mosquito-borne viruses, Semliki Forest virus (SFV) and the serotype 1 dengue virus (DEN1). We demonstrate that dsRNA can specifically inhibit transgene expression in C6/36 cells from both plasmid and SFV replicons and can significantly modify the kinetics of DEN1 RNA and virus replication. The inhibition mediated by dsRNA was sequence-specific and either equal or superior to that induced by antisense single-stranded RNA (ssRNA). This study demonstrates dsRNA-triggered inhibition of gene expression and virus replication in mosquito cells and suggests that this mechanism could be used to block pathogen replication within an insect host and, thus, block disease transmission.

Animals↗
PubMed2002

Gene therapy of HIV-1 infection using lentiviral vectors expressing anti-HIV-1 genes.

The use of vectors based on primate lentiviruses for gene therapy of human immunodeficiency virus type 1 (HIV-1) infection has many potential advantages over the previous murine retroviral vectors used for delivery of genes that inhibit replication of HIV-1. First, lentiviral vectors have the ability to transduce dividing and nondividing cells that constitute the targets of HIV-1 infection such as resting T cells, dendritic cells, and macrophages. Lentiviral vectors can also transfer genes to hematopoietic stem cells with a superior gene transfer efficiency and without affecting the repopulating capacity of these cells. Second, these vectors could be potentially mobilized in vivo by the wild-type virus to secondary target cells, thus expanding the protection to previously untransduced cells. And finally, lentiviral vector backbones have the ability to block HIV-1 replication by several mechanisms that include sequestration of the regulatory proteins Tat and Rev, competition for packaging into virions, and by inhibition of reverse transcription in heterodimeric virions with possible generation of nonfunctional recombinants between the vector and viral genomes. The inhibitory ability of lentiviral vectors can be further increased by expression of anti-HIV-1 genes. In this case, the lentiviral vector packaging system has to be modified to become resistant to the anti-HIV-1 genes expressed by the vector in order to avoid self-inhibition of the vector packaging system during vector production. This review focuses on the use of lentiviral vectors as the main agents to mediate inhibition of HIV-1 replication and discusses the different genetic intervention strategies for gene therapy of HIV-1 infection.

Genes, Viral↗
PubMed2002

Gene therapy-based treatment for HIV-positive patients with malignancies.

Gene therapy for the treatment of HIV has long been a goal of many investigators. The majority of trials have involved the use of lymphocytes transduced with vectors promoting resistance to HIV infection or replication. Unfortunately, the results have been less than encouraging with low-level marking and, more importantly, clearance of these lymphocytes from the circulation. Conversely, gene-modified hematopoietic stem cells appear able to introduce foreign transgenes while avoiding immunologic clearance. Furthermore, the use of less toxic conditioning regimens for allogeneic transplantation provides an attractive approach to conferring HIV resistance while allowing treatment of HIV-related disorders such as malignancies. This combination of nonmyeloablative allogeneic transplantation using gene-modified hematopoietic stem cell theoretically overcomes the high transplant mortality associated with traditional conditioning regimens in patients with HIV as well as providing a self-renewing source of HIV-resistant cells. To assess the safety and feasibility of such an approach, a clinical protocol was initiated in those patients infected with HIV with a hematologic malignancy meeting the standard indications for allogeneic transplantation and provided here is an update to the previously published original report. Only patient 1 received genetically modified cells. Both patients tolerated the procedure with no effect on viral load and improved CD4 counts, and patient 1 remains in complete remission from acute myelogenous leukemia 3 years post transplant. Patient 2 also achieved clinical remission from chemorefractory Hodgkin's disease but died of relapsed disease 12 months after transplantation. Vector-transduced cells remain detectable at low levels more than 3 years post-transplantation, suggesting the potential for gene therapy as a reasonable goal for the treatment of HIV.

CD4-Positive T-Lymphocytes↗
PubMedDate not supplied

Transduction of an HLA-DP4-restricted NY-ESO-1-specific TCR into primary human CD4+ lymphocytes.

cDNAs encoding functional T cell receptor (TCR) alpha and beta chains from a CD4+ T cell line (SG6) generated by repeated stimulation of a melanoma patient's peripheral blood mononuclear cells with HLA-DP4-restricted, NY-ESO-1-specific peptide p161-180 were cloned using a 5'rapid amplification of cDNA end method. Three different TCR alpha chains and 7 TCR beta chains were found among the 84 alpha and 162 beta cDNA clones tested. By screening different combination of the alpha/beta chains using RNA electroporation, TRAV9-1 (Valpha22.1) and TRBV20-1 (Vbeta2) were found to be the functional pair in line SG6. Antibody blocking experiments confirmed that the specificity of TRAV9-1/TRBV20-1 mRNA-transfected T cells were CD4 dependent and HLA-DP4 restricted. A retroviral vector expressing both TRAV9-1 and TRBV20-1 was constructed and used for transduction of OKT3-stimulated peripheral blood lymphocytes from melanoma patients. TCR-transduced CD4 T cells were capable of recognizing peptide-pulsed antigen-presenting cells (Epstein-Barr virus transformed B-cells, dendritic cells, and peripheral blood mononuclear cells), and protein-pulsed dendritic cells. Transduced cells were also capable of proliferation upon peptide stimulation and recognized peptide concentrations that were recognized by the parental line (0.2 microM). In contrast to SG6, which could not recognize human tumors, TCR-transduced CD4 T cells could specifically recognize NY-ESO-1/HLA-DP4-expressing melanoma cells. Major histocompatibility complex class II TCR-transduced CD4 T cells provides an alternative source of tumor antigen-specific T cells for adoptive immunotherapy of cancer patients.

CD4-Positive T-Lymphocytes↗
PubMedDate not supplied

Presentation of tumor antigens by dendritic cells genetically modified with viral and nonviral vectors.

Genetic modification of dendritic cells (DCs) with recombinant vectors encoding tumor antigens may aid in developing new immunotherapeutic treatments for patients with cancer. Here, we characterized antigen presentation by human DCs genetically modified with plasmid cDNAs, RNAs, adenoviruses, or retroviruses, encoding the melanoma antigen gp100 or the tumor-testis antigen NY-ESO-1. Monocyte-derived DCs were electroporated with cDNAs or RNAs, or transduced with adenoviruses. CD34+ hematopoietic stem cell-derived DCs were used for retroviral transduction. Genetically modified DCs were coincubated with CD8+ and CD4+ T cells that recognized major histocompatibility complex class I- and class II-restricted epitopes from gp100 and NY-ESO-1, and specific recognition was evaluated by interferongamma secretion. Cytokine release by both CD8+ and CD4+ T cells was consistently higher in response to DCs modified with adenoviruses than cDNAs or RNAs, and maturation of DCs after genetic modification did not consistently alter patterns of recognition. Also, retrovirally transduced DCs encoding gp100 were well recognized by both CD8+ and CD4+ T cells. These data suggest that DCs transduced with viral vectors may be more efficient than DCs transfected with cDNAs or RNAs for the induction of tumor reactive CD8+ and CD4+ T cells in vitro and in human vaccination trials.

Adenoviridae↗