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Biomedical subjects

G J McGarrity

Publications and source records attributed to G J McGarrity.

At least 19 recordsLinked to original sources
PubMed2005

Gene therapy progress and prospects: reprograming gene expression by trans-splicing.

The term 'trans-splicing' encompasses several platform technologies that combine two RNA or protein molecules to generate a new, chimeric product. RNA trans-splicing reprograms the sequences of endogenous messenger mRNA or pre-mRNA, converting them to a new, desired gene product. Trans-splicing has broad applications, depending on the nature of the sequences that are inserted or trans-spliced to the defined target. Trans-splicing RNA therapy offers significant advantages over conventional gene therapy: expression of the trans-spliced sequence is controlled by the endogenous regulation of the target pre-mRNA; reduction or elimination of undesirable ectopic expression; the ability to use smaller constructs that trans-splice only a portion of the gene to be replaced; and the conversion of dominant-negative mutations to wild-type gene products.

Animals↗
PubMed1999

In vitro analysis of transformation potential associated with retroviral vector insertions.

While replication-defective retroviral vectors provide excellent vehicles for the long-term expression of therapeutic genes, they also harbor the potential to induce undesired genetic changes by random insertions into the host genome. The rate of insertional mutagenesis for retroviral vectors has been determined in several different assay systems; however, the rate at which such events induce cellular transformation has not been directly determined. Such measurements are critical to determining the actual risk of carcinogenesis resulting from retroviral gene therapy. In this study, the ability of a replication-defective retroviral vector, GlnBgSvNa, to induce cellular transformation in the BALB/c-3T3 in vitro transformation assay was assessed. The transformation frequency observed in vector-transduced BALB/c-3T3 cells, which contained one to six copies of integrated provirus, was not significantly different from that of untreated control cells. The finding that GlnBgSvNa was nontransforming in this assay indicates that the rate of transformation induced by retroviral insertions is less than the spontaneous rate of cellular transformation by BALB/c-3T3 cells, or less than 1.1 x 10(-5). These results are the first to define an upper limit for the rate of transformation induced by retroviral vectors.

3T3 Cells↗
PubMed1999

Distribution of retroviral vectors and vector producer cells using two routes of administration in rats.

The clinical use of retroviral vector producer cells (VPCs) to deliver retroviral vectors efficiently to target cells has been investigated as a method to increase efficiency of gene delivery, presumably as a result of continued vector production in vivo. Studies were conducted in rats to evaluate the distribution of vector to distal organs and tissues as measured by transduction. Rats were treated with two doses of VPCs using two routes of administration: (1) subcutaneous injection, chosen to maximize both the dose and exposure of animals, thereby enabling identification of potential target organs under worst-case conditions; and (2) direct injection into brain parenchyma, chosen to mimic the intended clinical route of administration and provide an estimate of risk to patients receiving this therapy. Twelve organs or tissues were collected 7 days after administration of VPCs and analyzed by PCR for the presence of vector and vector producer cell sequences. Vector was detected most frequently at the site of injection by either route of administration. Less frequently, vector was detected in draining lymph nodes at the higher dose only using either route of injection. Single specimens of lung and contralateral skin were positive for vector following subcutaneous administration only. Vector was detected in gonadal tissue from a single low-dose male following subcutaneous administration, but this finding was not reproduced in any high-dose male or any males injected intracerebrally. In contrast, VPCs were detected only at the site of administration. The frequency of detection of VPCs 7 days after administration was higher when rats were injected by the intracerebral route. Based on these studies, gene transfer to distal organs or gonadal tissue following intracerebral administration of VPCs is not considered to be a risk to patients undergoing retroviral vector gene therapy for the treatment of brain cancer (glioblastoma multiforme; GBM).

Animals↗
PubMed1999

Efficacy of antiretroviral agents against murine replication-competent retrovirus infection in human cells.

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.

Cell Line↗
PubMed1998

Successful peripheral T-lymphocyte-directed gene transfer for a patient with severe combined immune deficiency caused by adenosine deaminase deficiency.

Ten patients with adenosine deaminase deficiency (ADA-) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA- severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA-SCID.

Adenosine Deaminase↗
PubMed1997

Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells.

Intratumoral implantation of murine cells modified to produce retroviral vectors containing the herpes simplex virus-thymidine kinase (HSV-TK) gene induces regression of experimental brain tumors in rodents after ganciclovir (GCV) administration. We evaluated this approach in 15 patients with progressive growth of recurrent malignant brain tumors. Antitumor activity was detected in five of the smaller tumors (1.4 +/- 0.5 ml). In situ hybridization for HSV-TK demonstrated survival of vector-producing cells (VPCs) at 7 days but indicated limited gene transfer to tumors, suggesting that indirect, "bystander," mechanisms provide local antitumor activity in human tumors. However, the response of only very small tumors in which a high density of vector-producing cells had been placed suggests that techniques to improve delivery and distribution of the therapeutic gene will need to be developed if clinical utility is to be achieved with this approach.

Adult↗
PubMed1995

Effect of herpes simplex virus thymidine kinase expression levels on ganciclovir-mediated cytotoxicity and the "bystander effect".

Transfer of the herpes simplex virus type-1 thymidine kinase (HSV-tk) gene into tumor cells followed by ganciclovir (GCV) administration, will provide selective tumor cell killing. We studied the effect of herpes simplex virus thymidine kinase (HSV-tk) expression level on the HSV-tk/GCV-mediated "bystander effect." Clones of HSV-tk-transduced rat glioma cells (9L) were isolated that stably expressed with different levels of HSV-tk. All clones studied had similar sensitivity to ganciclovir with IC50 values ranging from 0.45 to 1.3 microM. Within certain enzyme level thresholds, in vitro evaluation of the bystander effect has shown that clones with higher level of HSV-tk expression exhibited a better bystander effect. Interestingly, the bystander effect was observed between different cell types. Both the transduction efficiency and bystander effect are essential factors for the success of the antitumor effect by the HSV-tk/prodrug GCV suicide gene system.

3T3 Cells↗
PubMed1995

An improved retroviral vector encoding the herpes simplex virus thymidine kinase gene increases antitumor efficacy in vivo.

Brain tumors have been treated clinically by intratumoral injection of cells that produce retroviral vectors encoding the herpes simplex virus thymidine kinase (HSV-TK) gene followed by systemic administration of the antiviral drug ganciclovir. In vitro and in vivo comparisons of two different HSV-TK vector producer clones, which were made using standard transfection and transinfection techniques, were conducted. The two clones, PA317/G1TkSvNa.53 (TK.53) and PA317/G1Tk1SvNa.7 (TK1.7), both used in clinical trials, differ with respect to sequences 3' to the HSV-TK stop codon. The retroviral construct used to generate the TK.53 vector producer cell clone contains an open reading frame encoding a portion of the herpes simplex virus glycoprotein H (gH), a potential polyadenylation site and a putative splice site in this region. These sequences were removed from the retroviral construct used to create the TK1.7 vector producer cell clone. Supernatants obtained from TK1.7 vector producer cells had 100- to 1000-fold higher titers (G418 or HAT) than did corresponding supernatants from TK.53 vector producer cells. A murine subcutaneous tumor model was used to assess transduction efficiency and antitumor activity of each vector producer cell clone. In vivo tumor cell transduction was 13- to 18-fold more efficient with TK1.7 cells as compared with TK.53 cells at equivalent doses. Complete tumor ablation was achieved using a 10-fold lower dose of TK1.7 cells as compared with TK.53 cells. These results suggest that TK1.7 cells combined with ganciclovir may provide a more potent antitumor response in humans.

Animals↗
PubMed1994

Improved methods of retroviral vector transduction and production for gene therapy.

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.

3T3 Cells↗
PubMed1994

Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences.

A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.

Animals↗
PubMed1991

Elimination of mycoplasmas from cell cultures by a novel soft agar technique.

Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12-21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1-3 days. In separate studies, it was shown that certain Mycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas.

Agar↗
PubMed1991

Identification and characterization of proteinase K-resistant proteins in members of the class Mollicutes.

Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.

Animals↗
PubMed1990

Microbiological cultivation of Mycoplasma hyorhinis from cell cultures.

The failure of many cell culture isolates of Mycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly prepared yeast extract in mycoplasmal media together with incubation in 5% CO2/air successfully detected M. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures.

Cells, Cultured↗
PubMed1987

Activities of oxidative enzymes in mycoplasmas.

The activities of several oxidoreductases were measured in three fermentative and two nonfermentative Mycoplasma species that were grown under aerobic or anaerobic conditions. Acholeplasma laidlawii MG, Mycoplasma hyorhinis GDL, and Mycoplasma pneumoniae FH had very high apparent activities of pyruvate dehydrogenase and pyruvate dehydrogenase complex compared with the activities of mammalian fibroblasts or human platelet-enriched preparations, while Mycoplasma salivarium VV and Mycoplasma arthritidis 07 had very low apparent activities of these two enzymes. Strictly anaerobic growth diminished both enzymatic activities. The activity of alpha-ketoglutarate dehydrogenase complex was minimal in all five mycoplasmas that were grown under aerobic conditions, anaerobic conditions, or both. All the mycoplasmas that were examined exhibited lactate dehydrogenase and NADH-dichlorophenol indophenol oxidoreductase activities. The properties of mycoplasmal pyruvate dehydrogenase complex suggest that it differs from the mammalian enzyme.

Arsenic↗