Flanking-sequence exponential anchored-polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant-host-junction sequences.

BACKGROUND: Retroviral vectors are regularly used to transduce stem cells and their derivatives for experimental and therapeutic purposes. Because these vectors integrate semi-randomly into the cellular genome, analysis of integranated retroviral DNA/host cell DNA junctions (IHJ) facilitates clonality studies of engrafted cells, allowing their differentiation, survival and fate to be tracked. In the case of any adverse events, IHJ analysis can allow the identification of potentially oncogenic integration sites. At present, most measures to assess IHJ are complex, insensitive and may be subject to IHJ selection bias inherent to the technology used. METHODS: We have developed and validated a simple but effective technique for generating libraries of IHJ, which we term flanking-sequence exponential anchored-polymerase chain reaction (FLEA-PCR). Flanking-sequence random anchoring is used as an alternative to restriction enzyme digestion and cassette ligation to allow consistent detection of IHJ and decrease bias. RESULTS: Individual clones from plasmid libraries can be sequenced and assembled using custom-written software, and FLEA-PCR smears can be analyzed by capillary electrophoresis after digestion with restriction enzymes. DISCUSSION: This approach can readily analyze complex mixtures of IHJ, allowing localization of these sequences to their genomic sites. This approach should simplify analysis of retroviral integration.

Anaphylactic reaction after skin-prick testing in an 8-year-old boy.

This paper presents the case of an 8-year-old boy who developed an anaphylactic reaction after skin-prick testing (SPT). The tests were performed with commercial extracts and were strongly positive for dog and grass. The boy had no incidence of anaphylaxis reported in his history. At the time tests were performed, he had been admitted to the hospital because of persistent wheezing and had a dog at home for a few days. Although anaphylaxis is very rare after SPTs, these tests should always be peformed in a place equipped to treat anaphylaxis.

[Pica and iron-deficiency anemia in a 2-year old Indian child]. / Pica e anemia ferropriva in una bambina indiana di due anni.

Pica is the compulsive ingestion of nonnutritive substances. The causes are not known, but the symptom is often associated either with iron-deficiency or with pregnancy. The authors report on a case of pica in a 2-year old Indian child, affected by a serious ferropenic anaemia.

Influence of polarization mode dispersion value in dispersion-compensating fibers on the polarization dependence of Raman gain.

Polarization mode dispersion (PMD) of amplifying or transmitting fibers may significantly change the polarization dependence of Raman gain. Experimental results are presented that show low polarization dependence of the Raman gain in dispersion-compensating fibers with high PMD values for both copropagating and counterpropagating pumping configurations. The measured data demonstrate that it is possible to avoid the need for depolarization of a pump source in Raman amplifiers.

Bone marrow-derived cells contribute to tumor neovasculature and, when modified to express an angiogenesis inhibitor, can restrict tumor growth in mice.

Inhibition of tumor-induced neovascularization appears to be an effective anticancer approach, although long-term angiogenesis inhibition may be required. An alternative to chronic drug administration is a gene therapy-mediated approach in which long-term in vivo protein expression is established. We have tested this approach by modifying murine bone marrow-derived cells with a gene encoding an angiogenesis inhibitor: a soluble, truncated form of the vascular endothelial growth factor receptor-2, fetal liver kinase-1 (Flk-1). Murine bone marrow cells were transduced with a retroviral vector encoding either truncated, soluble Flk-1 (tsFlk-1) together with green fluorescent protein (GFP) or GFP alone. Tumor growth in mice challenged 3 months after transplantation with tsFlk-1-expressing bone marrow cells was significantly inhibited when compared with tumor growth in control-transplanted mice. Immunohistochemical analysis of tumors in each group demonstrated colocalization of GFP expression in cells staining with endothelial cell markers, suggesting that the endothelial cells of the tumor-induced neovasculature were derived, at least in part, from bone marrow precursors. These results suggest that long-term expression of a functional angiogenesis inhibitor can be generated through gene-modified, bone marrow-derived stem cells, and that this approach can have significant anticancer efficacy. Modifying these cells seems to have the added potential benefit of targeting transgene expression to the tumor neovasculature, because bone marrow-derived endothelial cell precursors seem to be recruited in the process of tumor-induced angiogenesis.

Autocrine expression of both endostatin and green fluorescent protein provides a synergistic antitumor effect in a murine neuroblastoma model.

Modalities that act through different mechanisms can often provide synergistic antitumor activity for the treatment of refractory tumors when used in combination. Here we report a gene therapy approach in which the genes for the angiogenesis inhibitor, endostatin, and the marker protein and potent immunogen, green fluorescent protein (GFP), were delivered to murine neuroblastoma cells prior to inoculation of the tumor cells into syngeneic immunocompetent mice. Although the effect of either angiogenesis inhibition or immunomodulation alone resulted in only a modest delay in tumor growth, when these approaches were used in combination, prevention of the formation of appreciable tumors was effected in 15 of 24 (63%) mice. The combination of endostatin and GFP expression elicited a strong immune response that was T cell-mediated and was reactive against both GFP and tumor cell line-specific antigens. This afforded treated mice protection against subsequent tumor challenge with unmodified tumor cells. These results suggest that antiangiogenic and immunotherapy strategies, when used in a gene therapy-mediated approach, can act synergistically in an effective multimodality anticancer approach.

Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukemic cells.

Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus 1.50 +/- 0.23 microM; P < 0.01). The lower sensitivity of TPMT+ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 +/- 282 versus 1515 +/- 183 pmol/5 x 10(6) cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 +/- 34 versus 27 +/- 10 pmol/5 x 10(6) cells; P = 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 +/- 0.6% versus 7.2 +/- 2.0%; P < 0.001). The higher sensitivity of TPMT+ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 +/- 1055 versus 174 +/- 77 pmol/5 x 10(6) cells; P = 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.

RD114-pseudotyped oncoretroviral vectors. Biological and physical properties.

Limited functional expression of the viral envelope receptor is a recognized barrier to efficient oncoretroviral mediated gene transfer. To circumvent this barrier we evaluated a number of envelope proteins with respect to gene transfer efficiency into primitive human hematopoietic stem cell populations. We observed that oncoretroviral vectors pseudotyped with the envelope protein of feline endogenous virus (RD114) could efficiently transduce human repopulating cells capable of establishing multilineage hematopoiesis in immunodeficient mice after a single exposure to RD114-pseudotyped vector. Comparable rates of gene transfer with amphotropic and GALV-pseudotyped vectors have been reported, but only after multiple exposures to the viral supernatant. Oncoretroviral vectors pseudotyped with the RD114 or the amphotropic envelopes had similar stability in vitro, indicating that the increased efficiency in gene transfer is at the receptor level likely due to increased receptor expression or an increased receptor affinity for the RD114 envelope. We also found that RD114-pseudotype vectors can be efficiently concentrated, thereby removing any adverse effects of the conditioned media to the long-term repopulating potential of the target human hematopoietic stem cell. These studies demonstrate the potential of RD114-pseudotyped vectors for clinical use.

Persistent low-level engraftment of rhesus peripheral blood progenitor cells transduced with the fanconi anemia C gene after conditioning with low-dose irradiation.

The hematopoietic stem cell has long been considered an ideal target for the introduction of therapeutic genes to treat human disorders such as Fanconi anemia (FA). Although recent progress in large animal models is encouraging, application to nonmalignant conditions is limited by the perceived necessity of myeloablative conditioning. We and others have shown that very low irradiation doses are sufficient to allow significant hematopoietic engraftment in murine hosts even after the introduction of xenogeneic genes. To determine the degree of engraftment of genetically modified cells attainable with very low irradiation doses in larger animals, we employed the rhesus macaque competitive repopulation model. Four animals underwent mobilization with stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) followed by apheresis. The apheresis product was enriched for the CD34-positive fraction by immunomagnetic selection and split equally for transduction with either G1FC26, a retroviral vector carrying the Fanconi anemia complementation group C gene, or PLII, a nonexpression control retroviral vector carrying both neomycin and beta-galactosidase gene sequences modified to prevent translation. Transductions were performed daily in the presence of fresh IL-3, IL-6, SCF, and Flt-3 ligand on fibronectin-coated plates over 96 h. Animals were conditioned with a single dose of either 100 (n = 2) or 200 (n = 2) cGy and received the combined products of transduction on the following day. None of the animals experienced clinically significant neutropenia nor required the use of central line placement, transfusional support with blood products, or intravenous antibiotics. Using real-time PCR, circulating levels of genetically modified cells as high as 1% were initially detected. Stable, albeit, significantly lower levels from both vector-transduced aliquots (<0.1%) persisted beyond 12 months posttransplant in all four animals. Although not sufficient to correct the phenotype in many human disorders, stable low-level engraftment by genetically modified cells following low-intensity conditioning may prove adequate in disorders such as FA due to the selective advantage conferred upon corrected cells.

Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA.

Long-term expression of coagulation factor IX (FIX) has been observed in murine and canine models following administration of recombinant adeno-associated viral (rAAV) vectors into either the portal vein or muscle. These studies were designed to evaluate factors that influence rAAV-mediated FIX expression. Stable and persistent human FIX (hFIX) expression (> 22 weeks) was observed from 4 vectors after injection into the portal circulation of immunodeficient mice. The level of expression was dependent on promoter with the highest expression, 10% of physiologic levels, observed with a vector containing the cytomegalovirus (CMV) enhancer/beta-actin promoter complex (CAGG). The kinetics of expression after injection of vector particles into muscle, tail vein, or portal vein were similar with hFIX detectable at 2 weeks and reaching a plateau by 8 weeks. For a given dose, intraportal administration of rAAV CAGG-FIX resulted in a 1.5-fold or 4-fold higher level of hFIX compared to tail vein or intramuscular injections, respectively. Polymerase chain reaction analysis demonstrated predominant localization of the rAAV FIX genome in liver and spleen after tail vein injection with a higher proportion in liver after portal vein injection. Therapeutic levels of hFIX were detected in the majority of immunocompetent mice (21 of 22) following intravenous administration of rAAV vector without the development of anti-hFIX antibodies, but hFIX was not detected in 14 immunocompetent mice following intramuscular administration, irrespective of strain. Instead, neutralizing anti-hFIX antibodies were detected in all the mice. These observations may have important implications for hemophilia B gene therapy with rAAV vectors.

Protection and in vivo selection of hematopoietic stem cells using temozolomide, O6-benzylguanine, and an alkyltransferase-expressing retroviral vector.

Transfer of drug resistance genes to hematopoietic stem cells offers the potential to protect cancer patients from drug-induced myelosuppression and to increase the number of gene-modified cells by in vivo selection. In this study, a retroviral vector expressing both a P140K variant of human O6-methylguanine-DNA methyltransferase (MGMT) and an EGFP reporter gene was evaluated for stem cell protection in a murine transplant model. Mice transplanted with vector-transduced cells showed significant resistance to the myelosuppressive effects of temozolomide (TMZ), an orally administered DNA-methylating drug, and O6-benzylguanine (BG), a drug that depletes cells of wild-type MGMT activity. Following drug treatment, increases in EGFP(+) peripheral blood cells were seen in all peripheral blood lineages, and secondary transplant experiments proved that selection had occurred at the stem cell level. In a second set of experiments in which transduced cells were diluted with unmarked cells, efficient stem cell selection was noted together with progressive marrow protection with repeated treatment courses. Altogether, these results show that P140K MGMT gene transfer can protect stem cells against the toxic effects of TMZ and BG and that this vector/drug system may be useful for clinical myeloprotection and for in vivo selection of transduced stem cells.

Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo.

BACKGROUND/PURPOSE: Preventing tumors from forming new blood vessels appears to be an effective new anticancer approach. Antiangiogenic therapy usually is cytostatic, however, and, therefore, long-term angiogenesis inhibition is likely to be required. The objective of this study was to determine if sustained gene therapy-mediated expression of these agents from tumor cells could restrict tumor growth in vivo. METHODS: Two replication-defective retroviral vectors were made, one encoding both the soluble, truncated vascular endothelial growth factor receptor (VEGF-R2), flk-1, together with green fluorescent protein (GFP), and the other encoding GFP alone. These vectors were then used to transduce murine neuroblastoma cells (NXS2). Stable, high expression of the flk-1 transgene was confirmed in the former population of cells by Western analysis. Flk-1 protein was isolated from cell culture supernatants and tested in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays to confirm that functional protein was being made. Finally, in vivo activity was assessed by injecting 10(6) tumor cells subcutaneously into SCID mice and monitoring subsequent tumor growth. RESULTS: Purified flk-1 (0.1 micromol/L) was able to inhibit basic fibroblast growth factor (bFGF) stimulated HUVEC proliferation by 44% and VEGF-stimulated migration by 30%. In vitro growth rates for the transduced cell lines were similar to the unmodified cell line. In vivo, however, after 23 days, tumors from flk-1 expressing neuroblastoma cells were less than 33% the average volume of tumors from cells expressing only the GFP transgene (mean volume, 1.9 cm(3) v 5.8 cm(3), P<.001). GFP expression alone had no effect on tumor growth when compared with unmodified tumor cells. CONCLUSIONS: Engineered expression of flk-1, a competitive inhibitor of VEGF, by tumor cells results in the production of an inhibitor of endothelial cell proliferation and migration that greatly restricts the growth of the tumor cells in vivo. Gene therapy-mediated delivery of angiogenesis inhibitors may provide an alternative approach to treating refractory tumors such as neuroblastoma.

Jak3 selectively regulates Bax and Bcl-2 expression to promote T-cell development.

Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.

The use of adenoviral vectors for genetic manipulation and analysis of primitive hematopoietic cells.

Gene transfer into stem cells has long been studied as a means by which primitive hematopoietic cells could be characterized and manipulated. While a variety of strategies have been attempted, it still remains relatively difficult to perform direct stem cell analysis. In this review, we examine recent studies using adenovirus-based vectors as a means to achieve high-level gene transfer into primitive hematopoietic cell types.

Retroviral vector-producer cell mediated angiogenesis inhibition restricts neuroblastoma growth in vivo.

BACKGROUND: The purpose of this study was to determine whether gene therapy-mediated delivery of an angiogenesis inhibitor, a truncated, soluble vascular endothelial growth factor receptor (Flk-1/KDR, VEGFR-2), could suppress tumor growth in a murine model of neuroblastoma. METHODS: Murine fibroblasts producing a replication-defective retrovirus encoding this mutant form of flk-1 were made. These producer cells were mixed with neuroblastoma cells and injected subcutaneously into SCID mice. Subsequent tumor growth was then measured. RESULTS: Murine neuroblastoma growth was decreased by 95% after 25 days. Similar tumor growth inhibitory effects were observed when the flk-1 producer cells were co-injected with cells from two different human neuroblastoma cell lines. CONCLUSIONS: Neuroblastoma growth can be significantly restricted in vivo with a single injection of cells that produce a retroviral vector encoding the gene for an angiogenesis inhibitor. This suggests that gene therapy-mediated delivery can be an effective alternative to chronic administration of these cytostatic anticancer agents.

Humoral response to vaccination with interleukin-2-expressing allogeneic neuroblastoma cells after primary therapy.

BACKGROUND: Immunotherapy using cytokine-expressing tumor cells has shown promise as an anticancer strategy. We have recently begun a trial of interleukin-2 (IL-2) gene-modified allogeneic neuroblastoma cells administered in a sequence of eight injections to patients with high-risk neuroblastoma following completion of primary therapy. Six patients to date have completed treatment. PROCEDURE: We examined humoral responses to the immunizing cell line and, when available, to the patients' autologous tumor cells using an in vitro binding assay. RESULTS: Five of six patients developed a rise in antitumor antibodies to the immunizing neuroblastoma cell line following vaccination. Two of these patients had autologous tumor available; both demonstrated a humoral response to these cells as well. CONCLUSIONS: Our results demonstrate that vaccination with IL-2-expressing allogeneic tumor cells after intensive primary therapy can elicit a humoral response to the immunizing line. These antibodies are cross-reactive with the patients' own tumor cells in the two cases in which autologous cells were available. This suggests that different patients' tumors may share common antigens that can be exploited in immunotherapy strategies and supports the continued exploration of allogeneic tumor cells as tumor vaccines.

Creation of polarized cells coexpressing CYP3A4, NADPH cytochrome P450 reductase and MDR1/P-glycoprotein.

PURPOSE: To develop model polarized cell systems expressing cytochrome P4503A4. NADPH P450 reductase, and P-glycoprotein (Pgp). METHODS: LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp, the product of the multidrug resistance gene (MDR1), were transfected stably using either a mammalian neomycin selectable expression vector (CYP3A4-Neo) or an episomal vector based on Epstein-Barr virus (CYP3A4-Hygro). These CYP3A4 expressing cells were compared with LLC-PK1, L-MDR1, or Caco-2 cells transduced with Adenovirus-3A4 vector (Ad3A4) with or without simultaneous Adenovirus-P450 Reductase (AdRed) transduction. Cells were characterized for expression of CYP3A4 protein and CYP3A4 mediated metabolism towards midazolam and testosterone. Analysis of membrane integrity and drug transport assays were performed to determine whether infection with recombinant Ad3A4 +/- AdRed affected Pgp function. RESULTS: The rank order of optimal CYP3A4 expression and activities in LLC-PKI and L-MDR1 cells from highest to lowest was cells cotransduced with Ad3A4 plus AdRed >> Ad3A4 >>> CYP3A4-Hygro > CYP3A4-Neo. Similarly, coexpression of Ad3A4 plus AdRed led to enhanced CYP3A4 mediated metabolism in Caco-2 cells over cells with Ad3A4 alone. Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detectable metabolite in the medium. In microsomes from Caco-2 and LLC-PK1 cells, each co-transduced with Ad3A4/AdRed, Vmax values for testosterone 6beta-hydroxylase activity ranged from 414 to 1350 pmoles/min/mg, respectively. For either Caco-2 or LLC-MDR1 cells, TEER values and the rate of apical to basal and basal to apical transport of vinblastine or digoxin were similar in cells with and without Ad3A4/Red transduction. CONCLUSIONS: Polarized cellular systems coexpressing Ad3A4, AdRed, and the MDR1/Pgp transporter were developed and characterized. The results document the utility of these polarized model systems for simultaneous drug transport/drug metabolism studies. Since the experimental approach can be adapted to study the interplay of multiple enzyme/ transporting systems, it may find significant application as a screening tool for the pharmaceutical industry and as a more basic research tool to study the kinetics of intestinal drug bioavailability.

Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.

Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector-mediated gene transfer. Human hematopoietic cell lines and cord blood-derived CD34(+) and CD34(+), CD38(-) cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34(+) cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive for enhanced green fluorescent protein represented as much as 90% of the graft. The use of RD114-pseudotyped vectors may be advantageous for therapeutic gene transfer into hematopoietic stem cells. (Blood. 2000;96:1206-1214)

Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN.

Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.