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Biomedical subjects

K W Culver

Publications and source records attributed to K W Culver.

36 records · Page 2Linked to original sources
PubMed1994

Clinical applications of gene therapy for cancer.

The number of gene therapy protocols for the treatment of cancer is growing rapidly. The most common type of approved clinical trial for cancer gene therapy involves the ex vivo gene transfer of cytokine genes (e.g., tumor necrosis factor, interleukin-2, granulocyte-macrophage colony-stimulating factor) into tumor cells. The idea behind this approach is to use gene transfer to induce a patient's tumor to become more immunogenic. The genetically altered tumor cells are reinjected into the patient in an effort to induce a systemic antitumor immune response against residual tumor cells. In other trials, investigators are using in situ gene transfer to selectively destroy cancer cells, sparing normal tissues. Continuing advances in molecular biology are likely to allow the development of new cancer treatments and methods of cancer prevention that will redefine cancer therapy.

Brain Neoplasms↗
PubMed1993

In situ retroviral-mediated gene transfer for the treatment of brain tumors in rats.

Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells. Rats inoculated with 4 x 10(4) 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (beta-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3-5 x 10(6) thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10(6)) resulted in a lower frequency of tumor regression (5 of 13 rats). To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10(6) beta-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranaside staining revealed maximal staining of beta-galactosidase within the tumor 7-14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4- chloro-3-indolyl-beta-D-galactopyranaside positive.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1993

Gene therapy for the treatment of brain tumors using intra-tumoral transduction with the thymidine kinase gene and intravenous ganciclovir.

Malignant brain tumors are responsible for significant morbidity and mortality in both pediatric and adult populations. These common tumors present an enormous therapeutic challenge due to their poor outcome despite radical surgery, high dose radiotherapy and chemotherapy. Survival of patients from the time of diagnosis is measured in months and recurrence after treatment is associated with a life expectancy of weeks. In an attempt to improve this grim prognosis of patients with malignant brain tumors (both primary tumors and secondary metastasis from systemic cancer such as melanoma, lung and breast cancer), we have developed a novel approach to the therapy of brain tumors. This approach makes use of recombinant DNA technology to transfer a sensitivity gene into a brain tumor. This is achieved by direct injection of the tumor with a cell line actively producing a retroviral vector carrying a gene conferring drug sensitivity to the tumor. A retroviral vector is a mouse retrovirus genetically engineered to replace its own genes with a new gene. Such vectors are capable of "infecting" mammalian cells and stably incorporate their new genetic material into the genome of the infected host. The producer cell is an NIH 3T3 cell that has been genetically engineered to continually produce retroviral vectors. The new gene is incorporated into the genome of the tumor cells and expresses the protein which is encoded by the new gene. This protein (the herpes simplex virus enzyme thymidine kinase, HS-tk) sensitizes the tumor cells to an antiviral drug (ganciclovir, GCV) which is a natural substrate for HS-tk. The enzymatic process induced by GCV leads to death of the cell expressing the herpes TK activity, i.e., death of the tumor cells. Since the HS-tk enzyme which is normally present in mammalian cells has very low affinity for GCV, systemic toxicity related to this mechanism is not observed. This type of in vivo gene transfer has several unique features. First, these retroviral-vectors will only integrate and express their genes in cells which are actively synthesizing DNA. Therefore, surrounding non-proliferating normal brain tissue should not acquire the HS-tk gene and will remain insensitive to GCV. Second, all of the transduced tumor cells (and retroviral vector producing cells) will be killed by the host immune response and/or GCV treatment eliminating potential concern about insertional mutagenesis giving rise to malignant cells. This is the first clinical attempt to treat malignant tumors in human beings by in-vivo genetic manipulation of the tumor's genome.

Adult↗
PubMed1993

Treatment of severe combined immunodeficiency disease (SCID) due to adenosine deaminase deficiency with CD34+ selected autologous peripheral blood cells transduced with a human ADA gene. Amendment to clinical research project, Project 90-C-195, January 10, 1992.

Significant increases in lymphocyte adenosine deaminase activity, T cell numbers and immune function have been achieved in the two children with SCID thus far treated with autologous T cells genetically-corrected by retroviral-mediated insertion of a normal ADA gene. Although the data obtained to date demonstrate that the use of ADA gene corrected peripheral T cells appears to be an effective treatment for ADA(-)SCID, it is theoretically preferable to try to develop a treatment for these children that will result in stem cell gene correction. The genetic correction of T cell progenitors with long-term immune reconstituting ability would be more desirable because repeated infusions of genetically altered cells should not be necessary and the generation of a more complete repertoire of T cell specificities might also be possible. Furthermore, the present treatment protocol involves indefinite continuation of enzyme replacement treatment with PEG-ADA. The demonstration of ADA gene expression in the progeny of transduced stem cells may simplify the decision concerning cessation of this very costly enzyme treatment (approximately $250,000/yr./patient). Recent evidence suggests that a small fraction of bone marrow or peripheral blood mononuclear cells bearing the CD34 antigen contains hematopoietic stem cells with both lymphoid and myeloid reconstituting ability. We propose in this amendment to supplement the infusion of human ADA gene-transduced autologous T cells in children with ADA(-)SCID with autologous peripheral blood CD34+ cells transduced with a second, readily distinguishable ADA vector.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Deaminase↗
PubMed1993

Toxicity studies of retroviral-mediated gene transfer for the treatment of brain tumors.

Retroviral-mediated transfer of the herpes simplex virus thymidine kinase (HSVtk) gene into malignant tumors confers drug susceptibility to the antiviral drug ganciclovir. The authors have recently shown that in situ transduction of the rat 9L brain tumor following HSVtk-producer cell implantation led to tumor regression after ganciclovir administration in treated rats. A wide spectrum of potential adverse effects may, however, be associated with the application of this approach to treat brain tumors, including dissemination of the retroviral vector to nontumoral tissues within or outside the central nervous system, proliferation of the injected murine vector-producer cells at the injection site, immune-mediated responses to the implantation of xenogeneic cells, and damage to the brain from toxic by-products of the HSVtk-ganciclovir interaction. These possibilities were investigated using intracerebral and systemic injections of retroviral vector-producer cells carrying the HSVtk or the lacZ gene in mice, rats, and nonhuman primates. Using the lacZ gene as a reporter gene, no evidence of beta-galactosidase activity consistent with vector transduction was detected in any major body organ in the treated mice or rats. Similarly, the HSVtk gene transfer did not result in toxicity, with or without ganciclovir administration. In studies using rat and monkey models, no proliferation of the vector-producer cells occurred after intracerebral injection. Vector-producer cell survival was limited to 7 to 14 days. High-dose steroid therapy did not appear to extend the survival of these xenogeneic cells in rats. No significant inflammatory response was observed in the meninges or brain parenchyma. Endothelial cells were occasionally transduced in brain capillaries adjacent to the injected site of the vector-producer cells. Injection of producer cells into brain tissue elicited mild edema and reactive gliosis surrounding the injection site, which were probably the cause of a transient toxic response arising 4 to 5 days following injection of the producer cells; short-term administration of dexamethasone eliminated that response. No neurological deficits were observed in the rats or primates treated with the HSVtk vector-producer cells, with or without ganciclovir. In primates injected with producer cells, magnetic resonance imaging before, during, and after ganciclovir administration showed minimal and localized breakdown of the blood-brain barrier without significant edema or mass effect. Similarly, histological examination of the monkeys' brains showed no damage to neurons, astroglia, or myelin. Long-term clinical (> 9 months) and radiological (3 months) assessment of the primates has revealed no evidence of toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors.

Direct in situ introduction of exogenous genes into proliferating tumors could provide an effective therapeutic approach for treatment of localized tumors. Rats with a cerebral glioma were given an intratumoral stereotaxic injection of murine fibroblasts that were producing a retroviral vector in which the herpes simplex thymidine kinase (HS-tk) gene had been inserted. After 5 days during which the HS-tk retroviral vectors that were produced in situ transduced the neighboring proliferating glioma cells, the rats were treated with the anti-herpes drug ganciclovir. Gliomas in the ganciclovir- and vector-treated rats regressed completely both macroscopically and microscopically. This technique exploits what was previously considered to be a disadvantage of retroviral vectors--that is, their inability to transfer genes into nondividing cells. Instead, this feature of retroviruses is used to target gene delivery to dividing tumor cells and to spare nondividing neural tissue.

3T3 Cells↗
PubMed1992

Gene therapy for primary immunodeficiency disease.

Gene therapy offers the potential for developing innovative new treatments for both inherited monogenic diseases as well as polygenic and acquired disorders. For most potential clinical applications, the technology has not yet progressed to the stage where it might be reasonably tested. Problems to be solved include the isolation and characterization of the genes involved, the development of gene delivery systems that will permit efficient gene insertion in the affected cells and tissues, and the development of mechanisms to control or appropriately regulate expression of the introduced genes. The primary immunodeficiency diseases as a group actually lend themselves to the development of gene therapy strategies with current technology more readily than almost any other class of disease. Theoretically any genetic disease that can be successfully treated by allogeneic bone marrow transplantation is a potential candidate for gene therapy directed at correcting the patient's own totipotent bone marrow stem cells. In addition, some disorders lend themselves to genetic correction of more mature cells, although gene transfer in this treatment strategy might have to be repeated periodically. The rationale and preliminary results of the first gene therapy protocol for ADA deficiency SCID are described and strategies for developing somatic cell gene therapy for the other primary immunodeficiency diseases are discussed.

Adenosine Deaminase↗
PubMed1991

Lymphocyte gene therapy.

Genetically corrected T cells are currently under investigation as a treatment for severe combined immunodeficiency disease resulting from a lack of adenosine deaminase (ADA). Monthly injections of these ADA-corrected T cells have resulted in measurable ADA activity in the peripheral blood and the in vivo production of antibody to blood group antigen. Genetically corrected T cells appear to be clinically valuable vehicles for gene therapy.

Adenosine Deaminase↗
PubMed1990

In vivo expression and survival of gene-modified T lymphocytes in rhesus monkeys.

Lymphocytes can be readily transduced with retroviral vectors and the gene-modified lymphocytes will stably express the inserted genes in vitro for long periods. As a prelude to studies in humans, we evaluated the survival of gene-modified T lymphocytes and the expression of the introduced genes in nonhuman primate T lymphocytes both in vitro and in vivo to determine if lymphocytes could be a potential cellular gene therapy vehicle. Rhesus peripheral blood T-lymphocytes and/or lymph node lymphocytes were transduced with a retroviral vector that contained a bacterial neomycin resistance (NeoR) gene or both NeoR and the human adenosine deaminase (hADA) genes. The cells were then selected for NeoR expression by growth in the neomycin analogue G418 and the autologous gene-modified T cells were reintroduced into the donor animals. T lymphocytes were periodically regrown from the blood and selected in G418. Gene-modified cells persisted in 1 animal for 727 days as detected by analysis for vector DNA by polymerase chain reaction (PCR). Evidence for expression of the human ADA or NeoR genes has also been detected up to 727 days after cell infusion. These findings suggest that gene-modified T lymphocytes can survive and circulate for long periods in vivo and can continue to express the introduced genes.

Adenosine Deaminase↗
PubMed1987

Lymphocyte abnormalities in infants born to drug-abusing mothers.

To evaluate the possible effects of maternal intravenous drug use on infant immunity, we measured the in vitro peripheral blood mononuclear cell proliferative responses to phytohemagglutinin (PHA) and pokeweed mitogen, T cell subset numbers, immunoglobulin levels, and titers of antibodies to cytomegalovirus (CMV) and human immunodeficiency virus (HIV) in a group of drug-abusing mothers and their infants. Infants of drug abusers had a lower proliferative response to mitogen, associated with altered kinetics of the maximum response to PHA. The OKT4/OKT8 ratio decreased with age in the drug-exposed infants compared with control infants (P less than 0.005). There was no evidence of CMV infection in either group. One mother and her infant had antibody to HIV. Our data demonstrate that infants of intravenous drug-using mothers have distinct immunologic differences at birth compared with non-drug-exposed infants and that these persist throughout the first year of life. The cause appears unrelated to intrauterine viral infection, suggesting a direct toxic effect of the drugs on fetal immunologic development.

Antibodies, Viral↗
PubMed1982

Children who set fires: the clinical picture and a follow-up.

The study concerns 46 children who had set at least one fire and had been admitted to a psychiatric ward. Their firesetting behaviour has been related to variables such as age, IQ, and psychiatric disorder in parents, and to the distinction between children who present with firesetting as their chief problem and those in whom it is a secondary complaint. Thirty subjects were followed up after one to five years. Seven, all boys and all less than 13 years old, were still setting fires, but these were less serious than the ones set before treatment. The persistent firesetters may have come from less stable homes and they tended to be more antisocial at follow-up than children who no longer set fires.

Adolescent↗