Search PubMed⌕ Search

Biomedical subjects

R A Morgan

Publications and source records attributed to R A Morgan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Genetic strategies to inhibit HIV.

The worldwide incidence of HIV infection continues to rise despite more than a decade of intense research aimed at developing effective intervention strategies. Because the mechanisms of action of the essential HIV gene products are now known, these have become potential targets for intervention. Some of these targets are attractive candidates for intervention by gene therapy. This review will focus on the recent progress in gene therapy strategies, including approaches approved for clinical trials. The efficacy of these various anti-HIV strategies, as well as the advantages and drawbacks of the different existing gene delivery systems, will be discussed.

Genetic Therapy↗
PubMed1999

Transplantation of transduced nonhuman primate CD34+ cells using a gibbon ape leukemia virus vector: restricted expression of the gibbon ape leukemia virus receptor to a subset of CD34+ cells.

The transduction efficiencies of immunoselected rhesus macaque (Macaca mulatta) CD34+ cells and colony-forming progenitor cells based on polymerase chain reaction (PCR) analysis were comparable for an amphotropic Moloney murine leukemia virus (MLV) retroviral vector and a retroviral vector derived from the gibbon ape leukemia virus (GaLV) packaging cell line, PG13. On performing autologous transplantation studies using immunoselected CD34+ cells transduced with the GaLV envelope (env) retroviral vector, less than 1% of peripheral blood (PB) contained provirus. This was true whether bone marrow (BM) or cytokine-mobilized PB immunoselected CD34+ cells were reinfused. This level of marking was evident in two animals whose platelet counts never fell below 50,000/microliter and whose leukocyte counts had recovered by days 8 and 10 after having received 1.7 x 10(7) or greater of cytokine-mobilized CD34+ PB cells/kg. Reverse transcriptase(RT)-PCR analysis of CD34+ subsets for both the GaLV and amphotropic receptor were performed. The expression of the GaLV receptor was determined to be restricted to CD34+ Thy-1+ cells, and both CD34+ CD38+ and CD34+ CD38dim cells, while the amphotropic receptor was present on all CD34+ cell subsets examined. Our findings suggest that, in rhesus macaques, PG13-derived retroviral vectors may only be able to transduce a subset of CD34+ cells as only CD34+ Thy-1+ cells express the GaLV receptor.

ADP-ribosyl Cyclase↗
PubMed1999

Gene therapy clinical trials for HIV.

Despite advances in drug treatment for human immunodeficiency virus (HIV), new therapeutics are needed for sustained treatment of this infection. Gene therapy has great potential for HIV due to its specificity for viral targets and prolonged expression of therapeutics. In this review, the various approaches that current gene therapy clinical trials are using to treat HIV disease are described. Results reported from these trials indicate that the approaches tested appear to be safe and confer some benefit to the genetically-modified cell. However, technical improvements must be made in order for gene therapy to realize its true potential.

Adoptive Transfer↗
PubMed1998

Ex vivo fibroblast transduction in rabbits results in long-term (>600 days) factor IX expression in a small percentage of animals.

Delivery of human factor IX to the circulation was analyzed in rabbits by ex vivo fibroblast transduction followed by subcutaneous implantation. Kinetic studies of human factor IX in rabbits demonstrated a half-life of approximately 16 hr and a volume distribution of 22%, where intraperitoneal and subcutaneous bioavailability was three- to sevenfold lower than by intravenous administration. Ex vivo retroviral transduction of autologous fibroblasts was performed on 15 animals. After subcutaneous injection of fibroblast-collagen mixtures, the expression of human factor IX in rabbit plasma was followed by ELISA. Of 15 rabbits injected, expression of human factor IX was detected in 2 animals, and expression was long term (>600 days). One animal had stable levels of human factor IX, at 20 ng/ml, while the second animal had lower and gradually decreasing levels of human factor IX. There were no gross differences in pathology at the injection sites, when comparing animals with human factor IX in plasma and those without. Immunological studies demonstrated antibody formation in response to injection mixture components (including human factor IX), but again there was no correlation with immune response and long-term factor IX production in animals. Tissues at the implantation sites were positive for factor IX DNA by PCR analysis, regardless of whether there was detectable plasma factor IX or not. Small numbers of PCR-positive cells were detected in the internal organs of the long term-expressing rabbits while similar tissues were negative in nonexpressing animals.

Animals↗
PubMed1998

Characterization of diverse viral vector preparations, using a simple and rapid whole-virion dot-blot method.

A number of different viruses have been adapted as gene transfer vectors, including retroviruses, adenoviruses, adenoassociated viruses (AAVs), herpes simplex virus, SV40 viruses, and alphaviruses (both Semliki Forest and Sindbis viruses). One of the major rate-limiting and time-consuming steps in the characterization of these vectors is the process of determining the viral vector titers. In addition, there is no "universal" method that can be used to rapidly estimate the titer and the utility of viral vector preparations. We demonstrate here that supernatant from diverse classes of viral vectors, with either RNA or DNA genomes, can be rapidly evaluated by a simple virus dot-blot hybridization without prior extraction of nucleic acids. This system can provide a reliable screen for physical titer of viral vector supernatants in 1 day.

Adenoviridae↗
PubMed1998

Targeted transduction of CD34+ cells by transdominant negative Rev-expressing retrovirus yields partial anti-HIV protection of progeny macrophages.

Congenitally acquired HIV infection may be uniquely suited to treatment via genetic engineering of CD34+ hematopoietic stem/progenitor cells. However, current technologies yield only a small percentage of mature cells that carry the inserted genes, and expression is frequently suppressed. Since clinical trials employing these methodologies have been proposed for anti-HIV gene therapy of HIV-infected children, we wished to assess, by in vitro modeling, the expected limits of transduction efficiency, expression, and antiviral activity using currently available methods. We measured retrovirus-mediated transduction in cord blood progenitors and their in vitro-derived progeny macrophages by Mo-MuLV vectors expressing a transdominant negative Rev (RevTD). CFU-GM transduction efficiency ranged from 7 to 85%, with an average of 28%. Semiquantitative DNA PCR demonstrated < or =100 vector sequence copies per 1000 cells in monocyte/macrophage cultures, which were grown without selection to better model in vivo conditions. When challenged with the macrophagetropic HIV-1BaL isolate, cultured macrophages from mock-transduced CFU-GM colonies supported infection in eight of eight experimental cultures, control LXSN-transduced progenitors supported infection in six of eight cultures, while macrophages derived from RevTD-transduced CFU-GM colonies supported infection in four of eight cultures. Although these results support the ability of neo(r) retroviral vectors containing RevTD to inhibit HIV replication, they indicate that further optimization of transduction efficiency and sustained expression will be required for effective anti-HIV protection in vivo.

Antigens, CD34↗
PubMed1998

Genetic induction of immune tolerance to human clotting factor VIII in a mouse model for hemophilia A.

Patients with severe coagulation factor VIII deficiency require frequent infusions of human factor VIII (hFVIII) concentrates to treat life-threatening hemorrhages. Because these patients are immunologically hFVIII-naive, a significant treatment complication is the development of inhibitors or circulating alloantibodies against hFVIII, which bind the replaced glycoprotein, increase its plasma clearance, and inhibit its activity, preventing subsequent treatments from having a therapeutic effect. A genetic approach toward the induction of immunologic unresponsiveness to hFVIII has the conceptual advantage of a long-term, stable elimination of undesired immune responses against hFVIII. Here, we report that in a factor VIII (FVIII)-deficient mouse model for severe hemophilia A, genetic modification of donor bone marrow cells with a retroviral vector encoding hFVIII, and transplant to hemophiliac mouse recipients, results in the induction of immune tolerance to FVIII in 50% of treated animals after immunization with hFVIII, despite the fact that hFVIII protein or activity is undetectable. In tolerized animals, the titers of anti-hFVIII binding antibodies and of hFVIII inhibitor antibodies were significantly reduced, and there was evidence for hFVIII unresponsiveness in CD4(+) T cells. Importantly, the plasma clearance of hFVIII was significantly decreased in tolerized animals and was not significantly different from that seen in a FVIII-naive hemophiliac mouse. This model system will prove useful for the evaluation of genetic therapies for hFVIII immunomodulation and bring genetic therapies for hFVIII tolerance closer to clinical application for patients with hemophilia A.

Animals↗
PubMed1998

Reduction in SIV replication in rhesus macaques infused with autologous lymphocytes engineered with antiviral genes.

Simian immunodeficiency virus (SIV) infection of nonhuman primates is one of the most relevant animals models of HIV infection in humans. To test a potential anti-HIV gene therapy strategy in this model, CD4-enriched lymphocytes from three rhesus macaques were subjected to retrovirally mediated gene transfer with a vector expressing an antisense tat/rev gene. This group of animals and three control macaques were subsequently infected with SIVmac239. Blood and lymph nodes from all macaques were sampled for more than a year to monitor the progress of infection. Although all animals became infected, the animals that received the lymphocytes engineered with the antisense vector demonstrated a significant reduction in viral load in both peripheral blood and lymph nodes, had sustained numbers of CD4+ cells, and exhibited little disruption of lymph node architecture.

Animals↗
PubMed1998

Gene therapy for infectious diseases.

Gene therapy is being investigated as an alternative treatment for a wide range of infectious diseases that are not amenable to standard clinical management. Approaches to gene therapy for infectious diseases can be divided into three broad categories: (i) gene therapies based on nucleic acid moieties, including antisense DNA or RNA, RNA decoys, and catalytic RNA moieties (ribozymes); (ii) protein approaches such as transdominant negative proteins and single-chain antibodies; and (iii) immunotherapeutic approaches involving genetic vaccines or pathogen-specific lymphocytes. It is further possible that combinations of the aforementioned approaches will be used simultaneously to inhibit multiple stages of the life cycle of the infectious agent.

Clinical Trials as Topic↗
PubMed1998

Development of improved adenosine deaminase retroviral vectors.

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a "simplified" vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA-) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA- severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.

Adenosine Deaminase↗
PubMed1997

Semliki Forest virus-mediated production of retroviral vector RNA in retroviral packaging cells.

Retroviral vectors are efficient tools for gene transfer studies. Their major advantage is that they can permanently integrate the transgene into the target cell's genome. However, because of the compulsory nuclear expression phase of their life cycle, it can be difficult for retroviruses to carry complex expression cassettes. In a attempt to mimic the structural features of most eukaryotic genes and obtain a potentially self-amplifying system for retrovirus production, we tested the feasibility of Semliki Forest virus (SFV) expression to mediate cytoplasmic synthesis of retrovirus vector RNA. An equivalent of a retrovirus virion RNA (retrovirus vector cassette, RVC) was cloned under the SFV 26S promoter, and full-length chimeric SFV-RVC RNA was produced in vitro. This RNA was introduced into retrovirus packaging cells, either via electroporation or transduction in SFV virions, and supernatants were analyzed for the presence of biologically active retroviruses. We demonstrate that this strategy can be used for cytoplasmic retrovirus production. The resulting viral particles are fully functional; they can transduce target cells, undergo reverse transcription, and integrate into genomic DNA. We also demonstrate that the SFV virion-based RVC delivery into packaging cells can yield high transient titers, in this case more than 10(5) G418R cfu/ml. This study shows that a simple, one-plasmid, heterologous viral RNA production system can be used to create functional retroviral RNA outside the cell nucleus.

3T3 Cells↗
PubMed1997

Retrovirus-mediated gene transfer of ornithine-delta-aminotransferase into keratinocytes from gyrate atrophy patients.

Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.

3T3 Cells↗
PubMed1997

Gut epithelial cells as targets for gene therapy of hemophilia.

Gut epithelium is an attractive target for gene therapy of hemophilia due to the large number of rapidly dividing cells that should be readily accessible to a wide range of vectors by a noninvasive route of administration. We have performed in vitro tests to determine the suitability of gut epithelial cells for gene transfer, protein synthesis, and secretion of coagulation factors VIII and IX. The results with retroviral vectors indicate that transduced epithelial cells from human, rat, or porcine small or large intestine can synthesize significant amounts of factor VIII or factor IX and that two-thirds or more of the recombinant protein is secreted in a basolateral direction (i.e., away from the lumen and toward underlying capillaries and lymphatics). Furthermore, we have demonstrated that intestinal epithelial cells are susceptible to efficient gene transfer by lipofection and adenovirus vectors. In the case of factor IX, we have produced a high-titer adenovirus vector capable of transducing gut epithelial cells resulting in synthesis of factor IX. The results of our in vitro studies indicate that gene transfer targeting gut epithelium as a new approach to hemophilia gene therapy is rational and merits in vivo studies in hemophilia animal models.

Adenoviridae↗
PubMed1997

A simple and reliable method for screening retroviral producer clones without selectable markers.

Simplified retroviral vectors that lack dominant selectable markers are being used with increasing frequency. These simplified vectors may offer a number of advantages over selectable marker-containing constructs, including potentially higher titers and less immunogenicity. However, the use of these vectors has been limited by the cumbersome experimental approaches in establishing and characterizing useful producer cell clones. To address this issue, a simple and reliable assay was developed to identify retroviral producer cell lines with or without dominant selectable markers. Producer cells were first generated by standard transfection/transduction and clones isolated by limiting dilution. Supernatant from each clone was then screened by RNA dot blot to identify the best producer clone candidates. The semiquantitative nature of the RNA dot blot assay was validated using a retroviral vector containing neomycin phosphotransferase (neo). Titers obtained by conventional G418-resistant colony forming units/ml (G418(R) cfu/ml) assays strongly correlated with the values by RNA dot blot procedure. RNA dot blot results also correlated well with titers estimated by Southern analysis of HeLa cells transduced with supernatant from each clone. The RNA dot blot technique is a rapid (2 days) and reliable method to screen retroviral producer cells, thereby facilitating the generation and characterization of simplified retroviral producer cell clones.

Animals↗
PubMed1997

In vivo retrovirus-mediated gene transfer into multiple hematopoietic lineages in rabbits without preconditioning.

Hematopoietic progenitor cells are attractive targets for gene therapy of inherited and acquired disorders. We have developed a novel procedure for mediating gene transfer into hematopoietic cells using an in vivo approach. The procedure involves injection of irradiated retroviral producer cells into the femoral bone marrow cavity in rabbits without preconditioning. The emergence of vector-marked cells in multiple peripheral blood hematopoietic lineages was detected 1 week post-injection and persisted until the animals were sacrificed up to 20 months later. Vector-marked cells were also detected in different hematopoietic tissues including bone marrow, spleen, thymus, and lymph node. Expression of retrovirus-specific messages by reverse transcription polymerase chain reaction was detected at multiple time points up to 20 months. Retrovirally encoded protein was detected by enzyme-linked immunosorbent assay in supernatant from cultures of granulocytes isolated 14 months after the procedure. This work demonstrates the feasibility of effecting gene transfer into hematopoietic progenitor cells in vivo.

Animals↗
PubMed1997

Efficient in vivo marking of primary CD4+ T lymphocytes in nonhuman primates using a gibbon ape leukemia virus-derived retroviral vector.

High efficiency retroviral-mediated gene transfer to rhesus CD4+ peripheral blood lymphocytes (PBL) was accomplished using an optimized transduction protocol using a gibbon ape leukemia virus (GaLV) envelope-containing packaging cell line PG13. Engineered CD4+ PBL were administered to three nonmyeloablated animals in three or four separate infusions over 9 months. Polymerase chain reaction (PCR) demonstrated in vivo reconstitution of the genetically engineered CD4+ PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene marking indicates that up to 30% of endogenous circulating CD4+ cells can be genetically engineered. The high levels of marked lymphocytes persist for the first 3 weeks following reinfusion then decline to < or = 0.1% over the next 21 weeks. Lymph node (LN) biopsies were performed to determine if the engineered CD4+ lymphocytes could traffic to lymphoid tissues. Marked lymphocytes were detected in LN biopsies 100 days following reinfusion of the transduced cells. Expression of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that were activated by culturing in the presence of recombinant interleukin-2 (rlL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expanded CD4+ lymphocytes. No antibody response was detected to the neomycin-resistance (Neo(R)) transgene, the murine retroviral group-specific antigen (gag), or GaLV envelope (env) proteins.

Animals↗
PubMed1997

Comparison of retroviral and adeno-associated viral vectors designed to express human clotting factor IX.

Several different designs for retroviral and adeno-associated virus (AAV) vectors were developed to express human clotting factor IX. Seven separate retroviral vectors were constructed, including chimeric long terminal repeat (LTR)-based designs, vectors containing splice donor/acceptor sites with internal ribosome entry sites (IRES), and vectors with an internal cytomegalovirus (CMV)- or hepatitis B virus (HBV)-derived promoter. Five AAV vectors were produced using the same cassette design where a viral promoter was used to transcribe a bicistronic mRNA containing factor IX and an IRES/neo gene. In the human hepatocyte cell line HepG2, the constructs were tested for factor IX production by ELISA, Northern blot, and Western blot, and for biological activity by normalization of the prolonged activated partial thromboplastin time (APTT) of factor IX-deficient plasma. All of the constructs produced biologically active factor IX in the range of 0.23-152 ng/24 hr per 10(6) cells (the HBV-promoted factor IX AAV vector was the least effective, and the CMV-promoted retroviral vector was the most active). Primary fibroblasts of both human and rabbit origin were also evaluated for factor IX production following transduction with viral vectors. Fibroblasts produced substantially more factor IX than the HepG2 cell line, with the best AAV vector synthesizing > 250 ng/24 hr per 10(6) cells and the best retroviral vector making > 900 ng/24 hr per 10(6) cells. Generally, we observed lower transduction efficiency and poorer expression with the AAV vectors versus retroviral vectors in these cell types.

Animals↗