Biomedical subjects

P S Obermiller

Publications and source records attributed to P S Obermiller.

10 recordsLinked to original sources

PubMed2000

Preclinical studies of a new generation retroviral vector for ovarian cancer BRCA1 gene therapy.

OBJECTIVE: The aim of this study was to determine the preclinical stability, toxicity, and efficacy of a second-generation complement-resistant retroviral BRCA1 vector, MFG-BRCA1, for ovarian cancer gene therapy. METHODS: MFG-BRCA1 was packaged in human 293 renal cells and manufactured and tested under cGMP conditions and is allowed for use in humans by the Food and Drug Administration. Vector stability studies were performed in mice and human serum by PCR analysis. Toxicity in the animals was assessed at necropsy, evaluating for histological signs of inflammation and organ damage. Tissue culture efficacy studies were performed on ovarian and breast cancer cells. Animal efficacy studies were conducted in female nu/nu mice. Mice were injected intraperitoneally with SKOV-3 ovarian cancer cells and tumors were allowed to grow for 4 weeks. Mice were treated intraperitoneally with MFG-BRCA1 or control vectors. Survival of animals was compared in the MFG-BRCA1 versus the control groups. RESULTS: MFG-BRCA1 was more stable in human serum than LXSN-BRCA1sv. Toxicity as demonstrated by an inflammatory peritonitis was minimal. Significantly fewer clones were obtained using the MFG-BRCA1 versus the MFG vector alone in both cell lines. Efficacy studies in animals of MFG-BRCA1 demonstrated a near threefold increase in survival over control vector and twofold increase compared to the first generation LXSN-BRCA1sv vector. CONCLUSION: The reengineered complement-resistant MFG-BRCA1 retroviral vector is more effective and more stable than the previous generation LXSN-BRCA1sv vector.

Animals↗

PubMed1999

Gene therapy for carcinoma of the breast: Therapeutic genetic correction strategies.

Gene therapy is a therapeutic approach that is designed to correct specific molecular defects that contribute to the cause or progression of cancer. Genes that are mutated or deleted in cancers include the cancer susceptibility genes p53 and BRCA1. Because mutational inactivation of gene function is specific to tumor cells in these settings, cancer gene correction strategies may provide an opportunity for selective targeting without significant toxicity for normal nontumor cells. Both p53 and BRCA1 appear to inhibit cancer cells that lack mutations in these genes, suggesting that the so-called gene correction strategies may have broader potential than initially believed. Increasing knowledge of cancer genetics has identified these and other genes as potential targets for gene replacement therapy. Initial patient trials of p53 and BRCA1 gene therapy have provided some indications of potential efficacy, but have also identified areas of basic and clinical research that are needed before these approaches may be widely used in patient care.

BRCA1 Protein↗

PubMed1999

Ovarian cancer BRCA1 gene therapy: Phase I and II trial differences in immune response and vector stability.

Gene therapy with viral vectors has shown some promise in nude mice models and in initial Phase I trials of patients with extensive metastatic cancer. A Phase I clinical trial (D. L. Tait et al., Clin. Cancer Res., 3: 1959-1968, 1997) of ovarian cancer patients treated with i.p. retroviral LXSN-BRCA1sv gene therapy reported stable vector, minimal antibody response, and tumor reduction. We initiated a Phase II trial on patients with less extensive disease to evaluate vector pharmacokinetics, immune response, toxicity, and efficacy. Patients received a surgically implanted peritoneal catheter to administer infusions of vector, as well as to retrieve daily samples of peritoneal fluid for analysis. Ovarian cancer patients received four daily i.p. injections of LXSN-BRCA1sv vector therapy for three cycles, 4 weeks apart. Patient peritoneal fluid and plasma were analyzed extensively by PCR, Western blot, complement level (CH50), and chemical and hematological tests. Phase II patients showed no response, no disease stabilization, and little or no vector stability. Because of vector instability and rapid antibody development, which differed dramatically from the Phase I trial data, the trial was terminated after treatment of six patients. Immune system status appears to have played a major role in whether gene therapy was effective. Comparison of Phase I and II patients showed significant differences in tumor burden, immune system status, and response to BRCA1 gene therapy.

Adolescent↗

PubMed1998

Ovarian cancer gene therapy.

Retroviral-mediated delivery of BRCA1 gene therapy (LXN-BRCA1sv, a normal splice variant form of BRCA1) was tested extensively in mouse models. It was found to be effective in reducing tumor burden and to be minimally toxic. Twelve phase I clinical trial patients with recurrent or persistent epithelial ovarian cancer were treated with one to three cycles of intraperitoneal vector. There was minimal toxicity, four patients developed fevers (< 102.5 degrees F) and three had sterile peritonitis, which resolved within 48 hours. The vector was found to be fairly stable in some patients at 24 hours as well as transferred into and expressed in patient tissues. Stable disease was noticed in 8 of the 12 patients, suggesting that the peritoneal cavity may be an appropriate site for gene therapy.

Animals↗

PubMed1997

A phase I trial of retroviral BRCA1sv gene therapy in ovarian cancer.

Gene transfer of BRCA1sv (a normal splice variant of BRCA1) into ovarian cancer cells produces growth inhibition in vitro and tumor suppression in nude mouse xenografts. As an initial step toward gene replacement therapy for ovarian cancer, we conducted a Phase I trial to assess the pharmacokinetics and toxicity of i.p. BRCA1sv retroviral vector therapy. Following placement of an indwelling Port-a-Cath in patients, a dose escalation study was performed of four daily i.p. infusions spanning doses from 3 to 300 ml (i.e., 10(10) viral particles) at half-log intervals (23 cycles in 12 patients). Gene transfer and expression were documented by PCR, Southern blot, reverse transcription-PCR, and nuclease protection assays. Pharmacokinetics were assessed by PCR and Southern blots detecting vector DNA, and toxicity was evaluated by clinical exam and fluid analysis. Three of 12 patients developed an acute sterile peritonitis, which spontaneously resolved within 48 h. Plasma and peritoneal antibodies to the retroviral envelope protein were detected only in patients treated with the highest dose levels but not in others, despite repeat dosing for an interval of up to 4 months. Eight patients showed stable disease for 4-16 weeks, and three patients showed tumor reduction with diminished miliary tumor implants at reoperation (two patients) and radiographic shrinkage of measurable disease (one patient). The vector-related complication of peritonitis was observed in three patients but resolved quickly as in preclinical mouse studies. Ovarian cancer may provide an important model for retroviral gene therapy studies due to vector stability, minimal antibody response, and access to tumor by i.p. therapy.

Adult↗

PubMed1995

Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression.

We have characterized expression of the familial breast and ovarian cancer gene, BRCA1, in cases of non-hereditary (sporadic) breast cancer and analyzed the effect of antisense inhibition of BRCA1 on the proliferative rate of mammary epithelial cells. BRCA1 mRNA levels are markedly decreased during the transition from carcinoma in situ to invasive cancer. Experimental inhibition of BRCA1 expression with antisense oligonucleotides produced accelerated growth of normal and malignant mammary cells, but had no effect on non-mammary epithelial cells. These studies suggest that BRCA1 may normally serve as a negative regulator of mammary epithelial cell growth whose function is compromised in breast cancer either by direct mutation or alterations in gene expression.

Alleles↗

PubMed1992

Activation-driven T cell death. II. Quantitative differences alone distinguish stimuli triggering nontransformed T cell proliferation or death.

Clonal deletion is the major mechanism by which T cell tolerance is achieved in vivo. The process of activation-driven cell death, originally characterized with T cell hybridomas, likely represents the mechanism of clonal deletion because it shares a number of properties with the in vivo process, especially the ability to be triggered in an Ag-specific manner, the cell-autonomous nature of the response, and its sensitivity to the drug cyclosporin A. We now have extended our analysis of activation-driven cell death to clonal populations of nontransformed T cells. Activation-driven cell death can be induced in nontransformed T lymphocytes by combinations of mitogenic stimuli. In particular, two mitogenic stimuli at high dose, one a lymphokine and the other delivered via the TCR or another activation structure, are required to induce activation-driven cell death. Activation-driven cell death is an active cell suicide process with attributes typical of physiological cell death, including early nuclear disintegration and a requirement for macromolecular synthesis, and is distinct from death by factor deprivation. Susceptibility to the induction of cell death by antigenic or activating stimulation is a common aspect of most T cells and is consistent with observations that clonal deletion can occur throughout T cell ontogeny. Most importantly, the alternative cellular responses of cell death and cell proliferation in nontransformed T cells appear to be triggered solely as a function of quantitative differences in the doses of identical stimuli. This can be viewed as a dose-dependent switch that determines cell fate. Developmental regulation of this switch may explain the processes of positive and negative selection during T cell ontogeny and also provide a mechanistic rationale for a strategy of selective anti-tumor therapy.

Animals↗

PubMed1992

Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes.

We examined virally transformed murine fibroblast clones as targets for cytotoxic T lymphocyte (CTL)-triggered lysis and genome digestion. Strikingly, while all clones were essentially equivalent in the ability to be lysed, one clone, SV3T3-B2.1, failed to exhibit genome digestion associated with CTL attack. Other aspects of the physiological cell death process, including loss of adhesion and nuclear envelope breakdown (lamin phosphorylation and solubilization), were not altered in this clone. The absence of genome digestion associated with CTL-induced cell death correlated with the absence of endodeoxyribonuclease activity in the nuclei of that clone. Characterization of the activity affected identifies a calcium-dependent, DNase I-like endonuclease of approximately 40 kDa, normally present constitutively in all cell nuclei, as the enzyme responsible for genome digestion associated with CTL-mediated cell death. These observations indicate that neither genome digestion per se nor its consequences [such as activation of poly(ADP-ribose) polymerase] are essential for cell death resulting from the triggering of this cell suicide process.

Animals↗

PubMed1990

Analysis of substrate specificity of the PaeR7 endonuclease: effect of base methylation on the kinetics of cleavage.

In murine cells expressing the PaeR7 endonuclease and methylase genes, the recognition sites (CTCGAG) of these enzymes can be methylated at the adenine residue by the PaeR7 methylase and at the internal cytosine by the mouse DNA methyltransferase. Using nonadecameric duplex deoxyoligonucleotide substrates, the specificity of the PaeR7 endonuclease for unmethylated, hemi-methylated, and fully methylated N6-methyladenine (m6A) and C5-methylcytosine (m5C) versions of these substrates has been studied. The Km, Kcat, and Ki values for these model substrates have been measured and suggest that fully or hemi-m6A-methylated PaeR7 sites in the murine genome are completely protected. However, the reactivity of fully or hemi-m5C-methylated PaeR7 sites is depressed 2900- and 100-fold respectively, compared to unmodified PaeR7 sites. The implications of the kinetic constants of the PaeR7 endonuclease for these methylated recognition sites as they occur in murine cells expressing this endonuclease gene are discussed.

5-Methylcytosine↗

PubMed1988

Introduction and expression of the bacterial PaeR7 restriction endonuclease gene in mouse cells containing the PaeR7 methylase.

To study the factors essential for a functional restriction system, the PaeR7 restriction-modification system has been introduced and expressed in murine cells. Transfer of this system was accomplished in two steps. First, cells containing sufficient PaeR7 methylase to completely methylate the mouse genome were constructed. In the second step, the mouse metallothionein promoter-regulated, endonuclease expression vector linked to the hygromycin B resistance selection marker was used to transfect the high methylase-expressing cells. Sixty percent of the clones isolated contained PaeR7 endonuclease enzymatic activity. Transfected cells expressing both methylase and endonuclease were incapable of blocking infection by DNA viruses, and possible explanations are discussed.

Adenoviridae↗