PubMed Health⌕ Search

Biomedical subjects

Richard A Morgan

Publications and source records attributed to Richard A Morgan.

14 recordsLinked to original sources
PubMed2004

Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells.

Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation. Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen. An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment. Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks). Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified. Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A.

Animals↗
PubMed2003

High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens.

The alpha- and beta-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-gamma), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4(+) T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.

Animals↗
PubMed2003

Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.

The use of lentiviral vectors for basic research and potential future clinical applications requires methodologies that can accurately determine lentiviral titers and monitor viral transgene expression within target cells, beyond the context of reporter genes typically used for this purpose. Here we describe a quantitative RT-PCR (qRT-PCR) method that achieves both goals using primer sequences that are specific for the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), an enhancer contained in many retroviral vectors and that is incorporated in the 3' UTR of nascent transgene transcripts. Quantitation of titers of three recombinant lentiviruses, genetically identical except for the transgene, demonstrated consistent differences in titer that were likely due to transgene-associated toxicity in producer cells and target cells. Viruses encoding the tumor-associated antigens tyrosinase and neo-poly(A) polymerase yielded reproducibly lower titers than a virus encoding enhanced green fluorescent protein (GFP) at the viral RNA, integrated DNA, and transgene mRNA levels, as measured by WPRE qPCR. Furthermore, the magnitude of differences in expression of the three transgenes in transduced target cells could not have been predicted by measuring vector DNA integration events. Since transgene expression in target cells is the most common goal of lentiviral transduction, and since methods to quantify transgene expression on the protein level are not always readily available, qRT-PCR based on a nucleotide sequence included in the transcript provides a useful tool for titering novel recombinant lentiviruses.

3' Untranslated Regions↗
PubMed2003

High-efficiency lentiviral vector-mediated gene transfer into murine macrophages and activated splenic B lymphocytes.

The goal of the present report was to determine if lentiviral vectors could mediate gene transfer into murine terminally differentiated macrophages and mature B lymphocytes as a new strategy of gene delivery into professional antigen-presenting cells (APC). We demonstrated that nondividing tissue resident macrophages were efficiently transduced in vitro by lentiviral vectors. Gene transfer efficiencies of up to 90% were demonstrated using a green fluorescent protein (GFP) reporter gene-containing vector and expression was stable for the length of cell culture. Transduced macrophages were functionally competent, preserving their phagocytic activity, accessory cell function, interleukin (IL)-12 secretion, and nitric oxide (NO) production similar to control untransduced macrophages. Lentiviral vector mediated transduction of CD19(+) B cell blasts was demonstrated to be in the range of 60%-70% GFP-positive cells. These transduced cells retain the ability to upregulate CD80 and CD86 similar to control B cell cultures. In addition, we show that the human immunodeficiency virus type 1 (HIV-1) accessory proteins Nef, Vpr, Vif, and Vpu are not required for the transduction of both resident macrophages and activated B lymphoblasts. We conclude that HIV-1-based lentiviral vectors can mediate efficient gene transfer into primary murine macrophages and mature B lymphocytes.

Animals↗
PubMed2002

The Chapel Hill hemophilia A dog colony exhibits a factor VIII gene inversion.

In the Chapel Hill colony of factor VIII-deficient dogs, abnormal sequence (ch8, for canine hemophilia 8, GenBank no. ) follows exons 1-22 in the factor VIII transcript in place of exons 23-26. The canine hemophilia 8 locus (ch8) sequence was found in a 140-kb normal dog genomic DNA bacterial artificial chromosome (BAC) clone that was completely outside the factor VIII gene, but not in BAC clones containing the factor VIII gene. The BAC clone that contained ch8 also contained a homologue of F8A (factor 8 associated) sequence, which participates in a common inversion that causes severe hemophilia A in humans. Fluorescence in situ hybridization analysis indicated that exons 1-26 normally proceed sequentially from telomere to centromere at Xq28, and ch8 is telomeric to the factor VIII gene. The appearance of an "upstream" genomic sequence element (ch8) at the end of the aberrant factor VIII transcript suggested that an inversion of genomic DNA replaced factor VIII exons 22-26 with ch8. The F8A sequence appeared also in overlapping normal BAC clones containing factor VIII sequence. We hypothesized that homologous recombination between copies of canine F8A inside and outside the factor VIII gene had occurred, as in human hemophilia A. High-resolution fluorescent in situ hybridization on hemophilia A dog DNA revealed a pattern consistent with this inversion mechanism. We also identified a HindIII restriction fragment length polymorphism of F8A fragments that distinguished hemophilia A, carrier, and normal dogs' DNA. The Chapel Hill hemophilia A dog colony therefore replicates the factor VIII gene inversion commonly seen in humans with severe hemophilia A.

Amino Acid Sequence↗
PubMed2002

Enhanced inhibition of human immunodeficiency virus type 1 replication by novel lentiviral vectors expressing human immunodeficiency virus type 1 envelope antisense RNA.

We have developed optimized versions of a conditionally replicating human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector for gene therapy of HIV-1 infection. These vectors target HIV-1 RNAs containing sequences of the envelope gene by expressing a 1-kb fragment of the HIV-1 Tat/Rev intron in the antisense orientation. Expression of the envelope antisense gene (envAS) was evaluated under the control of different internal promoters such as the human phosphoglycerate kinase (PGK) promoter, the human EF1-alpha promoter, and the U3 region of the SL3 murine leukemia virus. The U3-SL3 promoter transactivates transcription from the vector HIV-1 LTR and drives higher expression levels of envAS-containing RNAs than other promoters in T-cell lines. The effect of other vector structural features was also evaluated. We found that the central polypurine tract and central termination sequence (cPPT) produce a small increase in vector infectivity of 2-fold to 3-fold and results in a 10-fold higher inhibition of wild-type viral replication in challenge experiments. The woodchuck hepatitis posttranscriptional regulatory element (WPRE) does not increase the cytoplasmic levels of envAS mRNA in T-cell lines. We observed that SupT1 and primary CD4(+) T cells transduced with these vectors showed high inhibition of HIV-1 replication, suppression of syncitium formation, and increased cell viability when infected with several HIV-1 laboratory strains. Our results suggest that higher vector copy number and increased levels of envAS RNA expression contribute to block replication of divergent strains of HIV-1.

CD4-Positive T-Lymphocytes↗
PubMed2002

Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference.

RNA interference (RNAi) is a mechanism that appears to control unwanted gene expression in a wide range of species. In Drosophila, RNAi is most effectively induced by double-stranded RNAs (dsRNAs) of over approximately 80 nucleotides (nt) and in mammalian cells an RNAi-like inhibition of gene expression has been shown to be mediated by dsRNAs of approximately 21-23 nt. To test if RNAi can be used to specifically down-regulate a human disease-related transcript we have used Drosophila and human tissue culture models of the dominant genetic disorder spinobulbar muscular atrophy (SBMA). A variety of different dsRNAs were assessed for the ability to inhibit expression of transcripts that included a truncated human androgen receptor (ar) gene containing different CAG repeat lengths (16-112 repeats). In Drosophila cells, dsRNAs corresponding to non-repetitive sequences mediated a high degree of sequence-specific inhibition, whereas RNA duplexes containing CAG repeat tracts only induced gene-specific inhibition when flanking ar sequences were included; dsRNAs containing various lengths of CAG repeats plus ar sequences were unable to induce allele-specific interference. In mammalian cells we tested sequence-specific small dsRNAs of 22 nt; these rescued the toxicity and caspase-3 activation induced by plasmids expressing a transcript encoding an expanded polyglutamine tract. This study demonstrates the feasibility of targeting a transcript associated with an important group of genetic diseases by RNAi.

Animals↗
PubMed2002

Nonmyeloablative conditioning followed by transplantation of genetically modified HLA-matched peripheral blood progenitor cells for hematologic malignancies in patients with acquired immunodeficiency syndrome.

To assess the safety and efficacy of nonmyeloablative allogeneic transplantation in patients with HIV infection, a clinical protocol was initiated in patients with refractory hematologic malignancies and concomitant HIV infection. The results from the first 2 patients are reported. The indications for transplantation were treatment-related acute myelogenous leukemia and primary refractory Hodgkin disease in patients 1 and 2, respectively. Only patient 1 received genetically modified cells. Both patients tolerated the procedure well with minimal toxicity, and complete remissions were achieved in both patients, but patient 2 died of relapsed Hodgkin disease 12 months after transplantation. Patient 1 continues in complete remission with undetectable HIV levels and rising CD4 counts, and with both the therapeutic and control gene transfer vectors remaining detectable at low levels more than 2 years after transplantation. These results suggest that nonmyeloablative allogeneic transplantation in the context of highly active antiretroviral therapy is feasible in patients with treatment-sensitive HIV infection.

Acquired Immunodeficiency Syndrome↗
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↗