Sallie S. Boggs

Retroviral vectors for use in human gene therapy for cancer, Gaucher disease, and arthritis.

In the past several years, s igdcant advances have been made in the development of gene therapy for the treatment of certain human diseases such as severe combined immunodeficiency’ and cystic f ibr~s is .~ .~ Given these recent successes in gene therapy, researchers are now trying gene transfer as a therapy modality for a wide variety of genetic and acquired diseases. To this end, both viral and nonviral delivery systems have been developed, including retroviruses, adenoviruses, adenoassociated viruses, herpes simplex viruses, picornaviruses, and liposomes, some of which have been approved for use in clinical mals. Of these viral vector systems, retroviral vectors currently represent the best characterized vector and the most utilized delivery system for human gene therapy applications. This communication will discuss the use of retroviral vectors for gene therapy of neoplastic diseases, focusing on the use of a novel heterodimeric cytokine, interleukin (IL,)-12, for immunotherapy of cancer. In addition, the application of retroviral vectors for gene therapy for Gaucher disease, a well-characterized autosomal recessive lysosomal storage disease, and for an acquired autoimmune disease, rheumatoid arthritis, will also be discussed.

Comparisons of in vivo brdu labeling methods and spontaneous sister chromatid exchange frequencies in regenerating murine liver and bone marrow cells.

BrdU and BrdC have been employed as DNA labeling agents for differentiation of sister chromatids and for extension of sister chromatid exchange (SCE) methods to regenerating murine liver cells in vivo. Comparisons were made between bone marrow and liver cells isolated simultaneously from mice following DNA labeling with either BrdC or BrdU. Although the total mitotic yield of bone marrow cells was considerably greater than in liver, a higher percentage of second division metaphases was observed in liver cell preparations. The percentages of second division c-metaphase cells observed were 31.5% in bone marrow and 73% in liver cell preparations. Utilizing either BrdU or BrdC, no significant difference in percentage of second division metaphases was discerned. The number of spontaneous SCEs per cell was distributed according to the Poisson probability function. No significant differences in mean numbers of SCEs per cell were found in comparisons of bone marrow (1.40) and liver cells (1.65) or of cells which had incorporated BrdU or BrdC.

Lack of Natural Killer Cell Precursors in Fetal Liver of Ikaros Knockout Mutant Mice

The role of Ikaros in early stages of natural killer (NK) cell differentiation was investigated using an in vitro system that promotes proliferation and differentiation of NK cell precursors into mature NK1.1+ cells. Day 14.5 fetal liver cells from mice, either homozygous for Ikaros Null or dominant negative (DN) mutations, had severe 55- to 79-fold reductions in functional NK cell precursors. Although there was no statistically significant difference between values for +/+ and +/– Null mice, the mean precursor frequency for DN mutant (+/–) mice was significantly above that for DN –/– mice and below that for DN +/+ mice. The NK activity values for cells generated from the NK cell precursors followed the same respective relationships found for NK cell precursor frequencies. These data suggest that the deficiency of mature NK cells in Ikaros mutant mice is related to lack of functional precursors.

Reduced blood and marrow neutrophils and granulocytic colony-forming cells in sl/sld mice.

Summary The Sl/Sld mouse has been shown to have anemia and reduced megakaryocytes, but neutrophil levels in blood and marrow have not been studied previously to our knowledge. It proved to be neutropenic with reduced neutrophils and neutrophil precursors in marrow. The concentration of marrow cells forming granulocytic colonies in semisolid media was normal in young mice, but the total number per humerus was reduced. In vitro growth appeared normal in both semisolid and liquid culture, and peritoneal cells produced colony-stimulating activity normally. These results are compatible with previous studies indicating the Sl/Sld defect lies in the hematopoietic microenvironment rather than being a primary cellular defect, and suggest feedout from the pluripotent stem cell compartment may be reduced.

Effects of recombinant cytokines on colony formation by irradiated human cord blood CD34+ hematopoietic progenitor cells

The role of recombinant hematopoietic growth factors in radiation repair has become a subject of increasing interest in both clinical and basic radiobiology. Combinations of cytokines such as hepatocyte growth factor, interleukin (IL)-3, IL-11, kit ligand, GM-CSF and erythropoietin were used to study the in vitro radiation dose response of human cord blood CD34+ hematopoietic progenitor cells using clonogenic survival assays. CD34+ cells were isolated by immunomagnetic selection and irradiated at 8 cGy/min. Irradiated cells were plated in methylcellulose with or without added cytokines, and hematopoietic colonies including CFU-GM, BFU-E and CFU-GEMM were scored on day 14. The radiation response characteristics of BFU-E and CFU-GEMM were similar for all culture conditions tested. The D0 values for BFU-E ranged between 1.29 and 2.40 Gy and n between 1.0 and 1.4. The D0 values for CFU-GEMM ranged from 86 cGy to 2.02 Gy and n between 1.0 and 1.5. The D0 for CFU-GM grown without added factors was 1.03 Gy. With single cytokine stimulation (IL-3, IL-11 or varying concentrations of HGF), D0 values ranged from 1.11 to 1.44 Gy. With the combination of IL-3, GM-CSF, kit ligand and HGF, D0 values were not significantly altered and ranged between 1.61 and 2.60 Gy. In contrast, the combination of IL-11 and HGF produced an increase in the shoulder of the radiation survival curve (n = 3.35). No increase in the shoulder was detected for any of the other conditions tested (n = 1.0-1.7). Thus the combination of HGF and IL-11 increased the radiation survival of hematopoietic progenitor cells forming CFU-GM. Understanding the mechanism by which combinations of early-acting growth factors support postirradiation recovery of primitive clonogenic hematopoietic cells may be relevant to the design of clinical protocols for improving hematopoietic recovery after total-body irradiation.

Addition of serum to electroporated cells enhances survival and transfection efficiency

Optimal electroporation efficiency of many cell types is associated with poor survival. We show that serum rapidly reseals the membranes of electroporated cells and that timely addition of serum following electroporation can improve cell survival and transfection efficiency.

Polyamines in brain tumor therapy

SummaryIn the search for ways to augment current brain tumor therapies many have sought to exploit the fact that adult brain tissue is virtually lacking in cell division. This endorses a special appeal to therapeutic approaches which target the dependence on cell division for brain tumor growth. Polyamines play an essential role in the proliferation of mammalian cells and depletion results in inhibition of growth. As a result, there are investigations into the feasibility of controlling tumor growth by targeting the enzymes in polyamine metabolism with specific enzyme inhibitors. DFMO, an inhibitor of putrescine synthesis, is a cytostatic agent which in combination with tritiated radioemitters or cytotoxic agents such as, MGBG or BCNU is an effective antitumor agent, but the effectiveness of DFMOin vivo is reduced by tumor cell uptake of polyamines released into the circulation by normal cells and from gut flora or dietary sources. However, DFMO therapy combined with elimination of exogenous polyamines inhibits tumor growth but also results in body weight loss, reduced protein synthesis and evidence of toxicity. Furthermore, tumor growth recurs upon termination of treatment. In contrast, competitive polyamine analogs function in the homeostatic regulation of polyamine synthesis but fail to fulfill the requirements for growth and they continue to inhibit tumor growth for several weeks after cessation of treatment. Analogs are now in clinical trials. However, their action may be highly specific and differ from one cell type to another. We suggest that the effectiveness of polyamine based therapy would be enhanced by two approaches: local delivery by intracerebral microdialysis and tumor cell killing by internal radioemitters such as tritiated putrescine or tritriated thymidine which are taken up in increased amounts by polyamine depleted tumor cells. The growth inhibition by polyamine depletion prevents the dilution of the radioactive putrescine and thymidine. The overload of radioactivity kills the growth inhibited cells so that growth cannot recur when treatment terminates.

A new rat brain tumor model: Glioma disseminated via the cerebral spinal fluid pathways

SummaryA rat brain tumor model has been developed with the clinical and pathological features of dissemination via the cerebral spinal fluid (CSF) pathways. A precise number of 9L gliosarcoma cells (5 × 102 to 5 × 105) is stereotactically injected into the CSF of the lateral ventricle. The interval until the onset of neurological symptoms and then death is reproducible and dependent upon the number of cells injected. The median survival of three groups of rats receiving 5 × 105 cells in three different experiments was 17, 18 and 19 days respectively. For three groups receiving 5 × 104 cells, the median survival was 23, 24 and 25.5 days respectively and for two groups receiving 5 × 103 cells the median survival was 28 and 30 days respectively. The animals developed multiple tumor implants along the CSF pathways usually resulting in hydrocephalus. This tumor model was developed to simulate dissemination via CSF pathways as seen with medulloblastoma and other primitive neuroectodermal tumors of the central nervous system. It will be used to evaluate the therapeutic efficacy of intra-ventricularly administered anti-neoplastic drugs against small implants and malignant cells in the CSF pathways.

The Effect of Graded, Single Doses of Busulfan on Murine Erythropoiesis

Summary The effect of busulfan on murine erythropoiesis was studied following doses ranging from 10 to 100 mg/kg. The degree of depression of erythropoiesis was directly proportional to the dose of busulfan, as measured by hematocrit changes or by uptake of radioactive iron by spleen and marrow. The pattern of postbusulfan erythropoiesis was different from that seen after sublethal doses of whole body irradiation in some respects in that an abortive wave of erythropoiesis occurred in both marrow and spleen prior to onset of a more substantially sustained erythropoietic recovery. The pattern was like that following radiation in that the degree of reduction of erythropoiesis was directly related to dose as was the interval of time prior to onset of final recovery. In the spleen, this interval was 1 day per 20 mg/kg and in the marrow 0.5 days per 20 mg/kg.

Mouse Model for Assessing Endotoxin Involvement in the Lung Inflammation and Cytokine Production Resulting from Inhaled Organic Dust

AbstractThe involvement of endotoxin in the development of acute pulmonary inflammation and tumor necrosis factor (TNF) release following inhalation of cotton dust was demonstrated using endotoxin-sensitive C3HeB/FeJ and endotoxin-resistant C3H/Hel mice. These mice were exposed for a maximum of 6 h to atmospheres of either 45 mg/m3 cotton dust or 2.4 μg/m3 lipopolysaccharide (LPS) from Enterobacter agglomerans. Inflammation was assessed from bronchoalveolar lavage (BAL) cell morphology and lung histology. Release of TNF into BAL fluid was measured using a bioassay employing WEHI 13VAR cells and neutralization with rabbit anti-mouse TNF antiserum. Neutrophil influx and TNF release were maximal at 6 in C3HeB/FeJ mice following cotton dust exposure and at 3 h following LPS exposure. By 24 h after the beginning of cotton dust exposure, TNF in C3HeB/FeJ BAL was no longer detectable, whereas neutrophils were still elevated above control values. In endotoxin-resistant C3H/Hel mice, no inflammation or TNF release...