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R A Morgan

Publications and source records attributed to R A Morgan.

At least 19 recordsLinked to original sources
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↗
PubMed1997

Palliation of inoperable esophageal carcinoma: a prospective randomized trial of laser therapy and stent placement.

PURPOSE: A prospective, randomized comparison of the result of endoscopic laser therapy and that of placement of self-expandable metallic endoprostheses was performed to determine which method provides the best palliation of dysphagia in patients with inoperable esophageal carcinoma. MATERIALS AND METHODS: Sixty patients participated in the study. Twenty-three were randomly assigned to undergo plastic-covered stent placement, 19 to undergo uncovered stent placement, and 18 to undergo laser therapy. The quality of swallowing was assessed with the dysphagia score, which ranged from 0 for normal swallowing to 4 for complete dysphagia. RESULTS: The mean improvement in dysphagia score was 2 and ranged from -1 to 3 in patients who underwent placement of plastic-covered stents, was 2 and ranged from 0 to 4 in those who underwent placement of uncovered stents, and was 1 and ranged from 0 to 2 in those who underwent laser therapy. Six of 23 (26%) plastic-covered stents migrated, whereas none of the uncovered stents did so (P < .02). Tumor ingrowth through uncovered stents occurred in five of 19 patients (26%). CONCLUSION: Placement of metallic esophageal endoprostheses is substantially better than endoscopic laser therapy for palliation of dysphagia in patients with inoperable esophageal carcinoma. Use of uncovered and plastic-covered metallic stents provides equal palliation in patients with dysphagia.

Adult↗
PubMed1997

Gene therapy for HIV infection.

HIV-1 is the causative agent of acquired immune deficiency syndrome (AIDS) and is a major international health concern. The limited effectiveness of current antiviral therapies has led to the search for alternative treatments. One emerging field of medical treatment is termed human gene therapy. In principle, human gene therapy calls for the engineering of the cells from a medical patient with an agent that can potentially result in a therapeutic benefit to the patient. It has been suggested that gene therapy technology may be applied as an anti-HIV-1 agent. The term 'intracellular immunization' was suggested for strategies that work within a potential HIV-1 target cell to inhibit the productive infection of that cell by HIV-1. Various strategies based on this intracellular approach have been proposed and include the use of antisense, and transdominant HIV proteins. Both of these approaches can inhibit HIV-1 in vitro and a clinical trial has been proposed to test their effectiveness in vivo.

CD4-Positive T-Lymphocytes↗
PubMed1996

Retroviral gene transfer in autologous bone marrow transplantation for adult acute leukemia.

To evaluate whether marrow contributes to relapse after autologous bone marrow transplantation (AuBMT) for acute leukemia, transplanted marrow was marked with the G1N retroviral vector (Genetic Therapy Inc.) containing the neomycin phosphotransferase gene (neo). Between April 1992 and August 1993, 4 patients were transplanted for acute myeloid leukemia (AML) in second complete remission (CR) and 1 patient for acute lymphoid leukemia in first CR. An average of 12.4% (range 5-19%) of transplanted marrow mononuclear cells were exposed to G1N vector for 4 hr. In the vector-treated portion of the marrow, 4.9% of GM-CFU and 3.6% of erythroid burst-forming units (BFU-E) were resistant to G418 in vitro. In the 5 patients, the polymerase chain reaction (PCR) detected the neo sequence on only two occasions after AuBMT. Of 4 patients surviving 1 year after transplantation, only 1 had evidence of gene marked cells by PCR. Two AML patients have relapsed, one of whom had evidence of neo sequences in the bone marrow at day 100 but not at relapse 11 months after AuBMT. The second patient relapsed 18 months after AuBMT but never had PCR evidence of neo sequences before or after relapse. Our results indicate vector-transduced autologous bone marrow from heavily pretreated adults with acute leukemia mark with low efficiency, although vector sequences have been detected in bone marrow and peripheral blood up to 1 year after transplant. Of the 2 relapsed patients, no evidence of vector-marked leukemic blasts have been detected.

Acute Disease↗
PubMed1996

Molecular analysis of T lymphocyte-directed gene therapy for adenosine deaminase deficiency: long-term expression in vivo of genes introduced with a retroviral vector.

Peripheral blood lymphocytes from a patient with adenosine deaminase (ADA) deficiency were transduced in vitro with a replication-defective retroviral vector containing a human ADA-cDNA. Eighteen months after the last of a series of infusions of autologous retroviral vector-treated cells, vector sequences were detectable in DNA isolated from peripheral blood mononuclear cells (PBMCs), with an average copy number approaching one per cell. Increased ADA enzyme activity reaching approximately one-quarter normal levels was found in this population of cells. Other evidence of long-term retroviral vector expression in vivo included neomycin phosphotransferase (NPT) activity and demonstration of persistent vector mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). No evidence of spontaneous reversion of either mutant endogenous ADA allele was found.

Adenosine Deaminase↗
PubMed1996

Who's for TIPS?

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Catheterization, Central Venous↗
PubMed1995

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Adenosine Deaminase↗
PubMed1995

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

Animals↗
PubMed1995

Interleukin-2-transduced lymphocytes grow in an autocrine fashion and remain responsive to antigen.

The maintenance of T lymphocytes in vivo after adoptive transfer for immunotherapy requires the systemic administration of interleukin-2 (IL-2), but prolonged administration of IL-2 is associated with substantial toxicity. The constitutive production of IL-2 by T cells may be an alternative method to prolong T-cell survival and potentially augment antitumor responses. To study the effects of constitutive production of IL-2 on the growth and antigen reactivity of a murine T cell, the sperm-whale myoglobin (SWM) specific T-cell line 14.1 was retrovirally transduced with the cDNA for IL-2. Cells that were transduced with vectors without an internal promoter were able to proliferate in the absence of exogenously added IL-2, and to grow in an autocrine fashion. These vectors used an internal ribosomal entry site (IRES) to allow translation of the neomycin phosphotransferase (neor) gene. In contrast, the cells transduced with an IL-2 vector in which the neor gene was under the transcriptional control of an internal SV-40 promoter failed to proliferate or grow in the absence of exogenously added IL-2. The proliferation of the cells growing without IL-2 could be inhibited with antibodies to the IL-2 receptor or to human IL-2, indicating that they were still IL-2 dependent. Despite their autocrine growth, no tumor formation was observed in syngeneic mice injected subcutaneously with the transduced cells, and the cells retained their antigen reactivity and specificity. These results suggest that autocrine growth of T cells for therapy will not interfere with effector function.

Animals↗
PubMed1995

Multiple CTL specificities against autologous HIV-1-infected BLCLs.

The cellular immune response to HIV-1 has been well studied but, in many respects, remains incompletely defined. Although CTL specificities against highly conserved HIV-1 determinants as dictated by vaccinia/HIV-1 vector constructs have been described, much less is known regarding patient cellular reactivities against autologous cells infected with HIV-1. One of the main obstacles in characterizing this cellular reactivity has been the absence of a targeting system which accurately represents the HIV infected cell in vivo and is, at the same time, adaptable for in vitro assays. Through the use of two separate strategies aimed at increasing cellular CD4 expression, we were able to infect B-lymphocyte cell lines (BLCLs) with multiple strains of HIV-1. HIV-1-infected BLCLs were recognized by autologous effector cells with cytolytic specificities against env, gag, or pol determinants. In addition, HIV-1-infected BLCLs were capable of eliciting in vitro CTL reactivities directed against env-, gag-, and pol-expressing targets. This cellular reactivity was mediated by CD8+ cells and was MHC Class I restricted, suggesting a classical CTL response. Since multiple antigens are recognized, an HIV-1-infected BLCL is a more natural representation of an in vivo cellular target than other available testing systems and should permit a more representative analysis of CTL responses during infection or following vaccination.

B-Lymphocytes↗
PubMed1995

Analysis of trans-dominant mutants of the HIV type 1 Rev protein for their ability to inhibit Rev function, HIV type 1 replication, and their use as anti-HIV gene therapeutics.

The HIV-1 rev gene product facilitates the transport of singly spliced and unspliced HIV-1 transcripts and is necessary for productive HIV-1 infection. On the basis of the previously described trans-dominant Rev mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral expression vectors and analyzed for their ability to inhibit Rev-mediated gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-dependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expression was tested in the HeLa-T4 cell line and was also shown to be inhibited by the trans-dominant Rev mutants. Retroviral vector producer cell lines were constructed and used to transduce Rev trans-dominant genes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitored by Northern blot analysis and in situ hybridization. SupT1 cells expressing either a Rev point mutant or the frameshift mutant showed greatly reduced HIV-1 mRNA accumulation and the Rev-dependent singly spliced and unspliced HIV-1 mRNAs were reduced. The kinetics of viral replication following challenge of Rev trans-dominant-engineered SupT1 cells with both HIV-1 IIIB and MN strains was significantly reduced and cells were protected from viral lysis. Viruses that emerge late in infection from Rev trans-dominant-engineered cultures are not resistant to Rev-mediated inhibition. Last, trans-dominant Rev-mediated protection of human CD4+ lymphocytes from challenge with primary HIV-1 patient isolates confirms the potential utility of this system as an anti-HIV-1 gene therapy approach.

Amino Acid Sequence↗
PubMed1995

Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A.

To develop a potential gene therapy strategy for the treatment of hemophilia A, we constructed several retroviral vectors expressing a B-domain-deleted factor VIII (FVIII) cDNA. We confirmed previous reports that when the FVIII cDNA is inserted into a retroviral vector, the vector mRNA is decreased resulting in significantly (100- to 1,000-fold) lower vector titers. In an attempt to overcome this inhibition we pursued two independent strategies. First, site-directed mutagenesis was employed to change the structure of a putative 1.2-kb FVIII RNA inhibitory sequence (INS). Second, the FVIII gene was transcribed from a retroviral vector containing a 5' intron. Results demonstrated that the intron increased FVIII expression up to 20-fold and viral titer up to 40-fold but conservative mutagenesis of the putative FVIII INS region failed to yield a significant increase in FVIII expression or titer. Using the improved FVIII splicing vector, we transduced a variety of cell types and were able to demonstrate relatively high FVIII expression (10-60 ng of FVIII/10(6) cells/24 hr). These results underscore the usefulness of these transduced cell types for potential in vivo delivery of FVIII.

3T3 Cells↗
PubMed1995

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.

Acquired Immunodeficiency Syndrome↗