Non-Hodgkin's lymphoma in systemic lupus erythematosus.

BACKGROUND: Recent evidence supports an association between systemic lupus erythematosus (SLE) and non-Hodgkin's lymphoma (NHL). OBJECTIVES: To describe demographic factors, subtypes, and survival of patients with SLE who develop NHL. METHODS: A multi-site cohort of 9547 subjects with definite SLE was assembled. Subjects at each centre were linked to regional tumour registries to determine cancer cases occurring after SLE diagnosis. For the NHL cases ascertained, descriptive statistics were calculated, and NHL subtype frequency and median survival time of patients determined. RESULTS: 42 cases of NHL occurred in the patients with SLE during the 76,948 patient-years of observation. The median age of patients at NHL diagnosis was 57 years. Thirty six (86%) of the 42 patients developing NHL were women, reflecting the female predominance of the cohort. In the patients, aggressive histological subtypes appeared to predominate, with the most commonly identified NHL subtype being diffuse large B cell (11 out of 21 cases for which histological subtype was available). Twenty two of the patients had died a median of 1.2 years after lymphoma diagnosis. CONCLUSIONS: These data suggest aggressive disease in patients with SLE who develop NHL. Continuing work should provide further insight into the patterns of presentation, prognosis, and aetiology of NHL in SLE.

Children's emotional and behavioral disorders: attributions of parental responsibility by professionals.

In the wake of the neurobiological "revolution," do mental health professionals still assign etiological responsibility for emotional and behavioral disorders to deficient or harmful parenting? This study investigated differences in attributions of causality by theoretical orientation, professional discipline, areas of practice, familiarity with parent support groups, and demographic characteristics. Implications for policy, research, and practice are discussed.

Coexpression of rat glutathione S-transferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts.

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs.

Transient expression of Saccharomyces cerevisiae endo-exonuclease NUD1 gene increases the frequency of extrachromosomal homologous recombination in mouse Ltk- fibroblasts.

Endo-exonucleases (EEs) are nucleolytic enzymes which have been shown to participate in the processes of DNA repair and recombination in eukaryotes. Recently, we have demonstrated that transient expression of Saccharomyces cerevisiae EE NUD1 gene in HeLa cells increased the resistance of the latter to ionizing radiation and cisplatin, suggesting the involvement of the NUD1 gene product in the recombination repair of double-strand breaks (DSB). Here, we report that transient expression of NUD1 results in up to 62% increase in the frequency of homologous recombination between two co-transfected linear plasmids in mouse Ltk- cells.

Inhibition of poly(ADP-ribose)polymerase stimulates extrachromosomal homologous recombination in mouse Ltk-fibroblasts.

Poly(ADP-ribose)polymerase (PARP) is an abundant nuclear enzyme activated by DNA breaks. PARP is generally believed to play a role in maintaining the integrity of the genome in eukaryote cells via anti-recombinogenic activity by preventing inappropriate homologous recombination reactions at DNA double-strand breaks. While inhibition of PARP reduces non-homologous recombination, at the same time it stimulates sister chromatid exchange and intrachromosomal homologous recombination. Here we report that the inhibition of PARP with 100 microg/ml (0.622 mM) 1,5-isoquinolinediol results in an average 4.6-fold increase in the frequency of extrachromosomal homologous recombination between two linearized plasmids carrying herpes simplex virus thymidine kinase genes inactivated by non-overlapping mutations, in mouse Ltk-fibroblasts. These results are in disagreement with the previously reported observation that PARP inhibition had no effect on extrachromosomal homologous recombination in Ltk-cells.

The effect of the Saccharomyces cerevisiae endo-exonuclease NUD1 gene expression on the resistance of HeLa cells to DNA-damaging agents.

HeLa cells transiently transfected with a mammalian expression DNA vector expressing the Saccharomyces cerevisiae endo-exonuclease (EE) NUD1 gene have exhibited changes in cell survival frequencies after treatment with different DNA-damaging agents as compared to HeLa cells transfected with a control plasmid. The NUD1-transfected cells showed a dose-dependent increase in sensitivity to UV irradiation resulting in up to 58% decrease in cell survival. In response to gamma-irradiation NUD1 transfected cells featured an increased survival at doses equal to and greater than 2.0 Gy, reaching a maximum enhancement in survival frequency of 17%. At the same time, the NUD1-transfectants featured an increase in resistance to 0.25 microM-0.5 microM cis-platin (up to 58% increase in cell survival) and 1.0 mM EMS (11% increase). At higher concentrations of EMS NUD1 expression resulted in a decreased cell survival of the transfected cells (17% decrease for 2.5 mM EMS). No difference in cell survival frequencies between the NUD1-transfectants and the controls was observed after treatment with different concentrations of chlorambucil and mechlorethamine. These results suggest possible roles played by EEs in different DNA repair pathways--being stimulatory for the repair of certain types of DNA lesions, such as double strand breaks (DSBs), and interfering with the endogenous DNA repair systems for the repair of other types of lesions. Furthermore, these results also provide additional indirect evidence for the role of EEs in homologous recombination.

Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks.

The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination. The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes. While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death. Cell survival is inversely proportional to the duration of HO-endonuclease expression. Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression. On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient. Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52. Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within theMAT locus, suggesting that the survivors arose through the process of non-homologous end-joining. These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway.

Coexpression of cytidine deaminase and mutant dihydrofolate reductase by a bicistronic retroviral vector confers resistance to cytosine arabinoside and methotrexate.

The transfer of a drug resistance gene into hematopoietic cells is an approach being investigated to overcome the problem of myelosuppression produced by anticancer drugs. Chemotherapeutic agents are often given in combination in order to increase their effectiveness. Consequently, there is an advantage in designing vectors for gene transfer that are capable of expressing two drug resistance genes. We have constructed a bicistronic retroviral vector, MFG-DHFR-IRES/CD, which contains the mutated human dihydrofolate reductase (DHFR) cDNA with a phenylalanine-to-serine substitution at codon 31 (F31S) and the human cytidine deaminase (CD) cDNA. Murine fibroblast and hematopoietic cells were transduced with this vector and evaluated for their resistance to methotrexate (MTX) and cytosine arabinoside (ARA-C). The transduced fibroblast cells showed high levels of resistance to MTX and to ARA-C as determined by a clonogenic assay. Using enzymatic assays, we observed a coordinate increase in resistance to MTX and DHFR enzyme activity following an ARA-C selection. In addition, MTX selection produced an increase in CD enzyme activity and ARA-C resistance. Murine hematopoietic cells transduced with the bicistronic vector also showed drug resistance to both MTX and ARA-C. Interestingly, the double-gene construct conferred an equivalent level of drug resistance compared with single-gene vectors bearing only CD or DHFR genes in the hematopoietic cells. These results demonstrate the potential of the MFG-DHFR-IRES/CD vector to confer drug resistance to both MTX and ARA-C and may have future application in chemoprotection of normal hematopoietic cells in patients with cancer.

Drug resistance to 5-aza-2'-deoxycytidine, 2',2'-difluorodeoxycytidine, and cytosine arabinoside conferred by retroviral-mediated transfer of human cytidine deaminase cDNA into murine cells.

PURPOSE: The hematopoietic toxicity produced by the cytosine nucleoside analogs is a critical problem that limits their effectiveness in cancer therapy. One strategy to prevent this dose-limiting toxicity would be to insert a gene for drug resistance to these analogs into normal bone marrow cells. Cytidine (CR) deaminase can deaminate and thus inactivate 5-aza-2'-deoxycytidine (5-AZA-CdR), 2',2'-difluorodeoxycytidine (dFdC) and cytosine arabinoside (ARA-C). The aim of this study was to determine if gene transfer of CR deaminase into murine fibroblast cells confers drug resistance to these cytosine nucleoside analogs and if this resistance can be prevented by the CR deaminase inhibitor, 3,4,5,6-tetrahydrouridine (THU). METHODS: NIH 3T3 murine fibroblast cells were transduced with retroviral particles containing the human CR deaminase cDNA. Assays measuring CR deaminase activity as well as the inhibitory action of 5-AZA-CdR, dFdC and ARA-C on colony formation, were performed in the presence of different concentrations of THU. RESULTS: Retroviral-mediated transfer of the CR deaminase gene into 3T3 fibroblasts produced a considerable increase in CR deaminase activity. The transduced cells also showed significant drug resistance to 5-AZA-CdR, dFdC and ARA-C, as demonstrated by a clonogenic assay. This drug resistance phenotype and elevated CR deaminase activity were reversed by THU. CONCLUSIONS: These findings indicate that the CR deaminase gene can potentially be used in cancer gene therapy for protecting normal cells against the cytotoxic actions of different cytosine nucleoside analogs. In addition, the CR deaminase-transduced cells can be used as a model for screening different CR deaminase inhibitors in an intact cellular system.

Retroviral transfer and long-term expression of human cytidine deaminase cDNA in hematopoietic cells following transplantation in mice.

The chemotherapeutic effectiveness of cytosine nucleoside analogues used in cancer therapy is limited by their dose-dependent myelosuppression. A way to overcome this problem would be to insert the drug-resistance gene, cytidine deaminase (CD), into normal hematopoietic cells. CD catalyzes the deamination and pharmacological inactivation of cytosine nucleoside analogues, such as cytosine arabinoside (Ara-C). The objective of this study was to determine if we could obtain long-term persistence and expression of proviral CD in hematopoietic cells following transplantation of CD-transduced bone marrow cells in mice. Murine hematopoietic cells were transduced with an MFG retroviral vector containing CD cDNA and transplanted into lethally irradiated mice. The recipient mice were administered three courses of 10-15 h i.v. infusions of Ara-C (75-110 mg/kg). Blood, marrow and spleen samples were obtained and analyzed for CD proviral DNA by PCR, CD activity by enzyme assay, and drug resistance to Ara-C by clonogenic assay. We detected the presence of the CD proviral DNA in most of the samples examined. Approximately 1 year after transplantation several mice showed increased expression of CD activity in these tissues and some mice displayed signs of Ara-C resistance. These data demonstrate that persistent in vivo expression of proviral CD can be achieved in transduced hematopoietic cells and indicate some potential of this gene for chemoprotection to improve the efficacy of cytosine nucleoside analogues in cancer therapy.

Computer-aided breast cancer detection and diagnosis of masses using difference of Gaussians and derivative-based feature saliency.

A new model-based vision (MBV) algorithm is developed to find regions of interest (ROI's) corresponding to masses in digitized mammograms and to classify the masses as malignant/benign. The MBV algorithm is comprised of five modules to structurally identify suspicious ROI's, eliminate false positives, and classify the remaining as malignant or benign. The focus of attention module uses a difference of Gaussians (DoG) filter to highlight suspicious regions in the mammogram. The index module uses tests to reduce the number of nonmalignant regions from 8.39 to 2.36 per full breast image. Size, shape, contrast, and Laws texture features are used to develop the prediction module's mass models. Derivative-based feature saliency techniques are used to determine the best features for classification. Nine features are chosen to define the malignant/benign models. The feature extraction module obtains these features from all suspicious ROI's. The matching module classifies the regions using a multilayer perceptron neural network architecture to obtain an overall classification accuracy of 100% for the segmented malignant masses with a false-positive rate of 1.8 per full breast image. This system has a sensitivity of 92% for locating malignant ROI's. The database contains 272 images (12 b, 100 microm) with 36 malignant and 53 benign mass images. The results demonstrate that the MBV approach provides a structured order of integrating complex stages into a system for radiologists.

Resistance to cytosine arabinoside by retrovirally mediated gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells.

Dose-limiting hematopoietic toxicity produced by the cytosine nucleoside analogue cytosine arabinoside (ARA-C) is one of the major factors that limit its use in the treatment of neoplastic diseases. An interesting approach to overcome this problem would be to insert a gene for drug resistance to ARA-C in normal hematopoietic cells to protect them from drug toxicity. The deamination of ARA-C by cytidine deaminase results in a loss of its antineoplastic activity. The objective of this study was to determine if gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells would confer drug resistance to ARA-C. Retrovirally mediated transfer of the human cytidine deaminase gene into 3T3 fibroblasts resulted in efficient expression of the proviral RNA for this gene and in increased cytidine deaminase activity in cytoplasmic extracts. These cells showed marked resistance to ARA-C as determined by the effects of this drug on colony formation, cell growth, and DNA synthesis. The transfer of the human cytidine deaminase gene into murine bone marrow cells by the retroviral vector conferred a high level of drug resistance to ARA-C in clonogenic assays. These studies indicate that the cytidine deaminase gene could be used in cancer gene therapy by protecting normal hematopoietic cells against the cytotoxic effects of ARA-C and related cytosine nucleoside analogues.

Transfection of murine fibroblast cells with human cytidine deaminase cDNA confers resistance to cytosine arabinoside.

One of the major limitations in the use of cytosine arabinoside (Ara-C) in cancer chemotherapy is the hematopoietic toxicity produced by this nucleoside analog. One approach to overcome this problem would be to insert a gene for drug resistance to Ara-C in normal hematopoietic cells to protect them from drug toxicity. An interesting candidate gene for this aim is cytidine deaminase which catalyzes the deamination of Arac-C, resulting in a significant loss of its antineoplastic activity. We have ligated the human cDNA for cytidine deaminase into the plasmid vector pMFG. Transfection of NIH 3T3-derived GP + E86 murine fibroblasts cells with this vector resulted in a marked increase (> 50-fold) in the expression of cytidine deaminase. In addition, the transfected cells showed resistance to the cytotoxic action and to the inhibition of DNA synthesis produced by Ara-C. Northern and Western blot analysis of the transfected cells showed increased expression of mRNA for cytidine deaminase and increased immunologically detectable enzyme. The ability to confer drug resistance to Ara-C through gene transfer of cytidine deaminase may have the potential as a selectable marker and for the protection of the bone marrow from the toxicity produced by this analog so as to increase its effectiveness in cancer chemotherapy.

Retrovirus-mediated gene transfer of rat glutathione S-transferase Yc confers in vitro resistance to alkylating agents in human leukemia cells and in clonogenic mouse hematopoietic progenitor cells.

Recently, we have reported that N2Yc, a Moloney-based retrovirus vector expressing the Yc isoform of rat glutathione S-transferase (GST-Yc), conferred resistance to alkylating agents in mouse NIH-3T3 fibroblasts. In this report, we address the feasibility of using rat GST-Yc somatic gene transfer to confer chemoprotection to the hematopoietic system. Human chronic myelogenous leukemia K-562 cells were efficiently transduced with the N2Yc retrovirus vector and showed a significant increase in the 50% inhibitory concentration of chlorambucil (3.2- to 3.3-fold), mechlorethamine (4.7- to 5.3-fold), and melphalan (2.1- to 2.2-fold). In addition, primary murine clonogenic hematopoietic progenitor cells transduced with the N2Yc vector were significantly more resistant to alkylating agents in vitro than cells transduced with the antisense N2revYc vector. The survival of Yc-transduced hematopoietic colonies at 400 nM mechlorethamine and 4 mu M chlorambucil was 39.4% and 42.6%, respectively, compared to 27.2% and 30.4% for N2revYc-transduced cells. Future experiments will determine the level of chemoprotection achievable in vivo, following transplantation of N2Yc-transduced hematopoietic cells in mice.

Reliability of retrospective reports of perceived maternal acceptance-rejection in childhood.

Research reported here addresses the issue of the reliability of retrospective reports of children's perceptions of maternal acceptance-rejection as measured by the Parental Acceptance-Rejection Questionnaire. A sample of 49 middle-class Caucasian 7- to 11-yr.-old children were asked to respond to the questionnaire reflecting on their mothers' current accepting-rejecting behaviors. Seven years later the same children--now adolescents--responded to the same questionnaire with the instruction to reflect back on their mothers' behavior when the youth were about 7 to 11 years of age. None of these youth recalled having been tested seven years earlier. A simple zero-order correlation between scores in childhood and adolescence was 62, indicating that adolescents' retrospective recollections were in moderate agreement with their reports during childhood. Thus, it seems clear that, at least with respect to perceptions of maternal acceptance-rejection as measured by the Parental Acceptance-Rejection Questionnaire, researchers can have reasonable confidence that adolescents' current recollections about their experiences of maternal acceptance-rejection are likely to be in moderate agreement with what they would have reported had they been tested during childhood.

Gene therapy: here to stay.

Advances in biotechnology have brought gene therapy to the forefront of medical research. The feasibility of gene transfer was first demonstrated in experiments using tumour viruses. This led to the development of a variety of viral and nonviral methods for the genetic modification of somatic cells. Two main approaches emerged: in-vivo modification, in which gene transfer vehicles are delivered directly into patients, and ex-vivo manipulation, in which cells from the patient are grown in culture, genetically modified and then returned to the patient. In 1990, shortly after the safety of retrovirus-mediated gene transfer was demonstrated in patients with malignant melanoma, the first clinical trial of gene therapy was initiated for adenosine deaminase deficiency. Since then, the number of clinical protocols initiated worldwide has increased exponentially. Although some clinical trials now in progress are concerned with relatively rare inborn errors of metabolism, most are concerned with more commonly encountered cancers and infectious diseases. Preliminary results suggest that by the turn of the century the dream of treating diseases by replacing or supplementing the products of defective genes or introducing novel therapeutic genes will become a reality.

Retrovirus-mediated gene transfer of rat glutathione S-transferase Yc confers alkylating drug resistance in NIH 3T3 mouse fibroblasts.

A major limitation to successful cancer treatment is the existence of drug resistance. While several mechanisms of drug resistance have now been well characterized, mechanisms of resistance to alkylating drugs have remained less well defined. Several experimental models of alkylator resistance have implicated isoforms of glutathione S-transferase (GST) but transfection experiments using cloned isoforms of GST have yielded conflicting results. While there are several plausible explanations for these apparently contradictory findings, the issue that clonal variability might potentially confound the results of conventional transfection experiments has been raised. To address this issue properly, we have studied rat GST-Yc expression and drug sensitivity to alkylating drugs in populations of mouse NIH 3T3 fibroblasts following either transfection or transduction with an N2-based retrovirus vector. In comparison with cells treated with an antisense vector, Yc-transfected and Yc-transduced populations of NIH 3T3 cells expressed increased levels of GST-Yc mRNA (Northern blot), increased levels of immunodetectable GST-Yc (Western blot), and, respectively, 1.4- and 1.9-fold increases in total GST activity and 6.1- and 8.3-fold increases in glutathione peroxidase activity (associated with the Yc subunit). Yc-transfected and Yc-transduced cell populations were, respectively, 5.8- (P < 0.001) and 2.4-fold (P < 0.05) resistant to chlorambucil and 10.8- (P < 0.01) and 5.4-fold (P < 0.001) resistant to mechlorethamine. The range of resistance of clonal isolates from either population was 1.8-6.0-fold for chlorambucil and 4.6-6.1-fold for mechlorethamine (P < 0.05). In contrast, these cells showed unaltered sensitivity to the antimetabolite methotrexate, a nonalkylating drug. These results clearly demonstrate that the rat GTS-Yc is able to confer alkylating drug resistance in mouse fibroblasts. The ability to confer alkylating drug resistance following retrovirus-mediated gene transfer also raises the possibility of using GST-Yc somatic gene transfer to confer protection to the hematopoietic system in a gene therapy strategy applicable to cancer.

Pure red cell aplasia following ABO-incompatible bone marrow transplantation: response to erythropoietin.

Allogeneic bone marrow transplants with major ABO incompatibility may be associated with delayed erythroid engraftment. A case of a male patient with erythroleukemia (blood group O) who received a bone marrow transplant from an HLA-identical sibling (blood group AB) is reported. The bone marrow transplantation was followed by normal myeloid and megakaryocytic engraftment, but pure red cell aplasia was present for more than 230 days after bone marrow transplant. Despite documentation of an elevated endogenous erythropoietin level (360 mU/mL; normal value, < 19 mU/mL) during the period of absent erythropoiesis, erythroid engraftment was observed soon after the initiation of human recombinant erythropoietin at a dose of 50 U per kg daily. This experience suggests that high-dose erythropoietin may stimulate sufficient production of erythroid precursors to overcome circulating inhibitors resulting in the correction of pure red cell aplasia.

Gene therapy of the immune system.

Many applications of somatic gene therapy relate to the immune system. Several forms of inherited immunodeficiencies are candidates for treatment by gene transfer. Adenosine deaminase (ADA) deficiency causes a form of severe combined immunodeficiency. Stable gene transfer and expression of human ADA has now been obtained in hematopoietic stem cells of mice and, more recently, in large animals. The human ADA has also been introduced and expressed in the primitive human hematopoietic progenitor cells that initiate long-term bone marrow culture. Clinical trials of gene therapy for ADA deficiency have been initiated. The initial protocols were aimed at the correction of peripheral blood T lymphocytes, but recent strategies are attempting ADA gene transfer into peripheral blood or bone marrow stem cells. Other immunodeficiencies that may soon be amenable to somatic gene therapy include leukocyte adhesion deficiency and chronic granulomatous disease. Gene therapy may also be applied to the treatment of acquired disorders. In theory, the hematopoietic stem cells of a human immunodeficiency virus (HIV)-infected patient could be genetically modified and used to reconstitute an HIV-resistant hematopoietic system. Various strategies are currently being investigated to achieve this "intracellular immunization" against HIV. These include the transfer of genes encoding recombinant soluble CD4 molecules, suicide genes under the control of HIV-inducible promoter, and anti-HIV ribozymes. Gene transfer could also be used in the treatment of cancer to increase the immune response of the host, to activate prodrugs specifically in tumors, or to protect normal tissues against the toxicities of conventional treatment. Recent progress in all of these applications of gene therapy is reviewed here.

Characterization of recombinant helper retroviruses from Moloney-based vectors in ecotropic and amphotropic packaging cell lines.

We have characterized the recombinant replication-competent retrovirus (RCV) arising from p delta N2-derived vectors in the packaging cell lines psi 2 (ecotropic) and PA317 (amphotropic). Detailed restriction patterns and sequence of the envelope region of these RCVs has indicated that they arose from recombination events between the virus plasmids used to create the packaging cell line and the vectors. There was no evidence of recombination involving endogenous murine retroviral sequences in the packaging cell line or in transduced hematopoietic cells. In addition, we have confirmed that the mutation of the start codon of the pXM5(N2) derivatives gag+ sequence drastically decreased the occurrence of RCV production. These results offer encouragement that the risk of RCV production can be adequately decreased in gene therapy applications of defective retrovirus vectors.