Incorporation of drug resistance genes into gene vectors has 2 important roles in stem cell gene therapy: increasing the proportion of gene-corrected cells in vivo (ie, in vivo selection) and marrow protection to permit higher or more tightly spaced doses of chemotherapy in the treatment of malignant diseases. We studied in a clinically relevant canine model of gene therapy the P140K mutant of the drug resistance gene methylguanine methyltransferase (MGMT), which encodes a DNA-repair enzyme that confers resistance to the combination of the MGMT inhibitor O 6 -benzylguanine (O 6 BG) and nitrosourea drugs such as carmustine and methylating agents such as temozolomide. Two dogs received MGMT(P140K)-transduced autologous CD34 ؉ -selected cells. After stable engraftment, gene marking in granulocytes was between 3% and 16% in the 2 animals, respectively. Repeated administration of O 6 BG and temozolomide resulted in a multilineage increase in genemodified repopulating cells with marking levels of greater than 98% in granulocytes.MGMT(P140K) overexpression prevented the substantial myelosuppression normally associated with this drug combination. Importantly, hematopoiesis remained polyclonal throughout the course of the study. Extrahematopoietic toxicity was minimal, and no signs of myelodysplasia or leukemia were detected. These large-animal data support the evaluation of MGMT(P140K) in con IntroductionGene transfer to hematopoietic stem cells holds significant promise for the treatment of genetic diseases, AIDS, and cancer. The use of drug resistance genes in stem cell gene therapy has 2 important clinical applications. First, drug selection of genetically corrected cells in vivo increases the proportion of corrected cells in gene therapy protocols for genetic diseases affecting the hematopoietic system. This strategy could circumvent the relatively low gene transfer levels obtained after nontoxic reduced-intensity conditioning regimens. Second, this strategy could also be applied to protect the bone marrow in the context of chemotherapy for solid tumors. Chemoprotection could permit dose-intensified cancer chemotherapy regimens, averting the dose-limiting myelosuppression normally associated with intensified chemotherapy regimens. [1][2][3][4] Rescue with unmanipulated autologous stem cells has been widely used to this end, but this strategy allows for only a limited number of high-dose chemotherapy cycles and is still limited by short periods of significant myelosuppression in the period immediately after transplantation. In contrast, a genetically protected bone marrow could make possible tightly spaced dose-intense chemotherapy regimens over extended time periods in the complete absence of any myelosuppression.A particularly attractive drug resistance gene for use in gene therapy is the DNA-repair enzyme methylguanine methyltransferase (MGMT). 3,4 Overexpression of this enzyme renders primary hematopoietic cells resistant to nitrosoureas such as carmustine (BCNU) [3][4][5][6] and to methylating agents, such as t...
A wedge-shaped pattern of variation in stream fish standing stock estimates relative to a habitat variable, in which range of standing stocks increases as a function of the variable, is consistent with the concept that the habitat variable is a limiting factor for fish populations. This pattern of variation complicates interpretation of parameter estimates and significance of ordinary least-squares (OLS) regression models of conditional mean standing stock; slopes of these regression models may have little or no relation to slopes of models describing standing stock limits. We modeled standing stock limits by testing for homoscedastic error distributions, screening plots of coordinate pairs for evidence of a wedge-shaped pattern of data, and estimating 90th regression quantiles for simple linear models. Application of this technique to data sets supporting 35 previously published OLS regression models of stream fish standing stocks led to rejection of homoscedasticity (P < 0.10) in 13 of the 35 data sets. Eight of these heteroscedastic data sets had wedge-shaped patterns of variation in standing stock and slopes of 90th regression quantiles that differed from slopes of OLS regression models. For three of these eight data sets, tests rejecting homoscedasticity were more significant than tests rejecting zero slope parameters in OLS regression models. In a separate exercise, analysis of simulated standing stock data generated from known distributions indicated that our technique can detect heteroscedastic error distribution patterns and yield 90th regression quantile models of standing stock limits from data sets characterized by OLS regression as having no correlation between mean standing stock and a habitat variable. Identification of correlations between habitat variables and standing stock by OLS regression is a common method of determining whether a variable is to be used for habitat assessment. Application of our technique to data sets that display wedge-shaped patterns of variation should help identify variables that may be limiting standing stock from data sets that do not yield significant OLS regression models of mean standing stock.Numerous ordinary least-squares (OLS) regres-et al. (1988) indicate a continuing trend for greater sion models have been published that predict mean model precision to be associated with smaller data standing stock of fishes in streams from measures sets. All of the significant (P < 0.05) OLS models of macrohabitat variables such as stream width, described by Layher and Maughan (1985), Lanka average velocity, substrate composition, and total et al. ), Layher and Maughan (1987a, 1987b), dissolved solids. Fausch et al. (1988 reviewed 99 McClendon and Rabeni (1987), Pajak and Neves of these models and found that for small data sets (1987), Wesche et al. (1987), and Hubert and Rahel (especially those with less than 20 df) the models (1989) that had r 2 greater than 0.60 had 22 df or seemed to be precise predictors of mean standing fewer. stock and had coefficients of determinati...
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