Stable transduction of recombinant adeno-associated virus into hematopoietic stem cells from normal and sickle cell patients

The effectiveness of a health education programme for prevention of alcohol abuse and tobacco smoking among adolescents was assessed by an intervention study with a non-randomized control group. The study was carried out among 7th-9th grade school students in a Local Health Unit of the Brescia province, North Italy. Students were enrolled in either the intervention or the control group, based on the participation of their teachers to the educational programme. A total of 428 students were involved in the programme representing the intervention group, and 658 students not involved in the programme formed the control group. The health education programme included the prevention of alcohol abuse in the first school year (1989-90) and the prevention of tobacco smoking in the second year (1990-91). Each part of the programme included a series of lessons taken by the teachers for about 20 hours. Alcohol use and smoking habits, knowledge about, and attitudes toward alcohol and tobacco use of the students have been assessed by anonymous questionnaires. The questionnaires have been administered during school time at the beginning and the end of each school year, giving a total of 6 questionnaires, from Q1 to Q6. All the questionnaires included the same questions on alcohol and tobacco use, knowledge and attitudes. Of the 1086 students who attended the 7th grade in the year 1989-90, 428 were enrolled as intervention group and 658 as control group. As regards alcohol, the following findings were observed: 1. no difference was found in the percentages of students drinking at least one glass of wine or beer daily between the 2 groups at Q6; 2. no substantial change in the percentage of students aware of the health risks of alcohol abuse from Q1 to Q6 was observed. As regards tobacco smoking, the results of the study showed: 1. no difference between the 2 groups was found at Q6 as regards the proportion of students who smoked one or more cigarettes a month; 2. the percentages of students planning to smoke in the future were similar in the 2 groups in Q6; 3.the percentage of students with a high score on the health consequences of smoking was higher in the intervention than the control group at Q4, but again no difference between the groups was evident at Q6. In conclusion, the health education programme seemed to be unsuccessful in modifying behaviors and attitudes regarding alcohol and tobacco use during the 3-year follow-up.     

Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts: method and efficacy of adenovirus-mediated transduction

OBJECTIVE: Allograft-targeted immunosuppressive gene therapy may inhibit recipient immune activation and provide an alternative to systemic immunosuppression. We studied the optimal technique and efficacy of intracoronary gene transfer of viral interleukin-10 and human transforming growth factor-beta 1 in a rabbit model of heterotopic heart transplantation. METHODS: Replication-defective adenoviral vectors were constructed, expressing viral interleukin-10 (AdSvIL10) or transforming growth factor-beta 1 (AdCMVTGF-beta 1). Intracoronary delivery of vectors was accomplished ex vivo by either bolus injection or slow infusion. The allografts were implanted heterotopically in recipient rabbits and collected 4 days after the operation. Vector dose was 4 x 10(9) to 6 x 10(10) pfu/gm of donor heart. Transfer was confirmed by DNA amplification for both genes. Gene product expression in tissue was quantified by immunoassay and visualized by immunohistochemical staining. RESULTS: Allograft viral uptake was only 9.9% +/- 2.4% with bolus injection, but increased to 80.5% +/- 6.8% at 1 ml/min infusion rate (p = 5 x 10(-14)). Uptake ratio was not affected by vector quantity or slower infusion rates. Transforming growth factor-beta 1 was consistently detected in allografts infected with AdCMVTGF-beta 1, but not with control adenovirus or AdSvIL10. Expression was proportional to infused vector quantity and reached 10 ng/gm of allograft at infused 10(10) pfu/gm. Transforming growth factor-beta 1 was also detected in recipient's serum at less than 1 ng/ml. Viral interleukin-10 was detected in minor amounts only (< 1 ng/gm) in allografts infected with AdvIL10 up to 5 x 10(10) pfu/gm. Nevertheless, it was detected in recipient serum at concentrations up to 0.4 ng/ml. CONCLUSIONS: Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts during cold preservation is feasible. Slow infusion is superior to bolus injection. In vivo effects on allograft rejection remain to be determined.     

Animal experiment on gene therapy of ovarian cancer by adenovirus-mediated thymidine kinase gene transduction and ganciclovir administration in vivo

OBJECTIVE: The efficacy and toxicity of adenovirus-mediated transduction of herpes simplex virus thymidine kinase gene started by Rous sarcoma virus (ADV/RSV-tk) followed by administration of ganciclovir (GCV) were studied in vivo. METHODS: An animal model of human epithelial ovarian cancer was established in nude mice using the serous ovarian adenocarcinoma cell lines Ov-ca-2774, then mice were treated by ADV/RSV-Tk and GCV, or GCV and HSV-tk respectively. The average survival time of mice and toxicity were assessed. RESULTS: The mice treated with GCV or HSV tk alone died from 14.4 +/- 1.7 to 19.3 +/- 3.5 days after treatment. The survival time had no difference with control group. The mice treated with ADV/RSV-tk followed by GCV lived at least two times longer than controls and the difference in both groups was significant. The earlier the treatment began, the longer the average survival time was. Treatment efficacy was dependent on dose of ADV/RSV-tk and tumor burden of mice. CONCLUSION: ADV/RSV-tk gene therapy is a safe and efficient approach to ovarian cancer treatment in the experiment.     

Enhancement of adenovirus-mediated gene transfer to human bone marrow cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complexes

DNA separations which traditionally have been performed by slab gel or capillary electrophoresis, may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of mass measurements. We report here typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS. Allelic ladders, which are typically used with electrophoretic separation systems to correct for mobility differences of DNA fragments under various conditions, are not needed for accurate genotyping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to approximately 0.1 nucleotide, was routinely observed. Mass accuracies were better than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addition, the smaller PCR product sizes (55-125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic samples with degraded DNA.     

Efficient, repeated adenovirus-mediated gene transfer in mice lacking both tumor necrosis factor alpha and lymphotoxin alpha

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, and the related cytokine lymphotoxin alpha (LTalpha), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-alpha/LTalpha-/-), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-alpha/LTalpha-/- and TNF-alpha/LTalpha+/- mice versus TNF-alpha/LTalpha+/+ mice links antibody levels to TNF-alpha/LTalpha gene dosage. Due to the absence of neutralizing antibodies, the TNF-alpha/LTalpha knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-alpha in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.     

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.     

Cytotoxic effects of adenovirus-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells

To evaluate the effects of the wild-type p53 expression in normal and tumor cells, we have constructed a recombinant adenovirus vector (E1 minus) expressing human wild-type p53 cDNA (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWTp53 resulted in high levels of wild-type p53 expression. Production of p53 protein following infection was dependent on the dose of AdWTp53 with maximum amounts of p53 produced following infection with 50 plaque-forming units/cell. AdWTp53 infection inhibited the growth of all human cell lines studied. However, tumor cells that were null for p53 prior to infection (H-358 and MDA-MB-157) and tumor cells that expressed mutant endogenous p53 protein (MDA-MB-231 and MDA-MB-453) were more sensitive to AdWTp53 cytotoxicity than cells that contained the wild-type p53 (MCF-7, MCF-10, 184B5, and normal mammary epithelial cells). All cells exhibited WAF1/Cip1 mRNA and protein induction following AdWTp53 infection. AdWTp53-induced cytotoxicity of human tumor cell lines expressing mutant p53 was mediated by apoptosis as revealed by nucleosomal DNA fragmentation analysis. No detectable nucleosomal DNA fragmentation was observed following AdWTp53 infection of human cells expressing wild-type p53. These data suggest that endogenous p53 status is a determinant of AdWTp53-mediated cell killing of human tumor cells.     

Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy

Astrocytic tumors frequently express Fas/APO-1 (Fas), in sharp contrast to surrounding normal brain cells, providing a potential window through which selective killing of tumor cells could be pursued. To assess this possibility, we transduced Fas into U251, a glioma cell line resistant to anti-Fas antibody-mediated apoptosis, and obtained transfectants with high levels of Fas expression. Anti-Fas antibody showed significantly enhanced cytotoxicity for the transfectants, suggesting that U251 cells maintained an intercellular cascade of Fas-mediated apoptosis. When U251 transfectants with high-level Fas expression were transduced with Fas ligand-encoding gene via retrovirus, they were unaffected by exposure to anti-Fas antibody or Fas ligand adenovirus (Adeno-FL). Thus, retroviral induction of Fas ligand into the glioma cells with high levels of Fas led to the selection of cells that were resistant to Fas-dependent apoptosis. These resistant U251 transfectants were susceptible to FADD adenovirus (Adeno-FADD)-induced apoptosis, indicating that a cascade of death signals was blocked at the steps between Fas ligand and FADD. As for adenoviral transduction of Fas ligand into gliomas, gliomas with a relatively high level of expression of Fas were remarkably sensitive to Adeno-FL-induced apoptosis. Besides, Adeno-FADD induced pronounced apoptosis in all glioma cells. Our data suggest the possibility of using adenovirus-mediated transduction of Fas ligand and FADD genes as a therapeutic approach to target gliomas.     

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo, which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity.     

Retrovirus-mediated gene transfer into human hematopoietic stem cells

Activation and infection by HIV-1 of glial cells and infiltrating macrophages are cardinal features of AIDS-related neurological disease. Tumor necrosis factor-alpha (TNF-alpha) is released by these cell types, and increased TNF-alpha mRNA and protein levels are associated with the development and severity of HIV-induced neurological disease. HIV-1 proteins have been implicated in HIV neuropathogenesis including Tat which has been shown to be a potent inducer of TNF-alpha. We review our data showing the induction of TNF-alpha by Tat in primary human fetal astrocytes, human peripheral blood mononuclear cells, macrophages, and astrocytic and macrophage cell lines. TNF-alpha induction was NF-kappaB dependent and was eliminated by inhibiting protein kinase A, phospholipase C and protein tyrosine kinase activity. In addition, we examined the molecular diversity of the tat genome in the brains of HIV-infected patients from different HIV-1 clades. Comparison of matched brain- and spleen-derived tat sequences indicated that homology among brain-derived clones was greater than that between the brain- and spleen-derived clones. The brain-derived tat sequences were markedly heterogeneous in regions which influence viral replication and intracellular transport. Future studies using Tat, encoded by different sequences, will be necessary to determine the functional significance of tat molecular diversity. Nonetheless, these studies suggest that Tat is an important inducer of TNF-alpha production and thus may play a key role in the pathogenesis of HIV-related neurological disease.     

Thymosin beta4, inhibitor for normal hematopoietic progenitor cells

Thymosin beta4 (Tbeta4), isolated from the calf thymus fraction 5, has a ubiquitous localization and plays a pleiotropic role in both the immune and nonimmune systems. Because it contains at its N-terminal end the sequence of a known inhibitor of hematopoiesis, the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP, Goralatide), we have assayed Tbeta4 on human hematopoietic cells. We demonstrate that it inhibits normal bone marrow progenitor cell growth; indeed, it decreased the growth of both granulo-macrophagic and erythroid progenitors and reduces their percentage in S phase. Furthermore, we show that Tbeta4 reduces both the clonogenicity and the cell proliferation of purified CD34+ cells induced by a combination of seven growth factors. Although Tbeta4's inhibitory effect is very similar to that of AcSDKP, we demonstrate, using neutralizing antibodies and a truncated form of Tbeta4 devoid of the AcSDKP sequence, that the inhibitory effect of Tbeta4 is not mediated by the sequence AcSDKP. These data indicate that Tbeta4 is a novel inhibitor for human normal hematopoietic progenitors.     

Retroviral-mediated gene transduction of human alkyltransferase complementary DNA confers nitrosourea resistance to human hematopoietic progenitors

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy due to low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in myeloid precursors. We have shown that high-efficiency myeloproliferative sarcoma virus (vM5MGMT)-mediated transduction of the human MGMT cDNA into murine bone marrow (BM) cells leads to high MGMT expression and increased progenitor resistance to 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) in vitro immediately after infection and after BM transplantation. These experiments were designed to increase MGMT expression in human hematopoietic progenitors. CD34(+) BM cells were isolated over an immunoaffinity column (CEPRATE, CellPro, Inc.), resulting in a mean 66-fold enrichment in clonogenic progenitors (colony-forming unit granulocyte-macrophage + burst-forming unit erythroid + colony-forming unit granulocyte erythroid macrophage = megakaryocyte), with an average progenitor yield of 58 +/- 11.5% and a final population that was 54% CD34(+). Seventy % of progenitors derived from CD34(+) cells were transduced after coculture with AM12-vM5MGMT retroviral producers. vM5MGMT-transduced progenitors were over 2-fold more resistant to concentrations of BCNU between 30 and 50 micrometer than were concurrently LacZ-transduced progenitors (P < 0.003). In vitro selection of transduced, cytokine-stimulated CD34(+) cells with 20 micrometer BCNU resulted in survival of 4.7% of MGMT+ clonogenic progenitors compared to 0.05% of LacZ+ progenitors. These studies indicate that MGMT-transduced human hematopoietic progenitors have increased resistance to nitrosoureas, and in a clinical transplant setting, this strategy may reduce alkylating agent myelosuppression.     

Abrogation of TGF-beta activity during retroviral transduction improves murine hematopoietic progenitor and repopulating cell gene transfer efficiency

Transforming growth factor-beta has complex activities on hematopoietic cells. We have previously shown that murine long-term repopulating activity is compromised by ex vivo culture in TGF-beta 1 and conversely is increased by abrogating endogenous TGF-beta activity with a neutralizing antibody. In the current study, we investigated the effect of abrogation of autocrine or paracrine TGF-beta present during retroviral transduction on gene transfer efficiency to primitive hematopoietic cells. Murine marrow cells were cultured and retrovirally transduced for 4 days in the presence of interleukin-3, interleukin-6 and stem cell factor, and either a neutralizing anti-TGF-beta antibody or an isotype control. Committed progenitor cells were analyzed for gene transfer efficiency, and cells were also injected into W/Wv recipient mice for analysis of transduction of long-term repopulating cells. The progenitor (CFU-C) transduction efficiency in the presence of anti-TGF-beta was significantly greater. Semiquantitative PCR analysis and Southern blot analysis for the retroviral marker gene in the blood and bone marrow of recipient mice revealed a significant increase in the transduction efficiency of long-term repopulating cells after culture and transduction in the presence of the anti-TGF-beta. Thus neutralization of TGF-beta activity during retroviral transduction allows more efficient gene transfer into primitive murine hematopoietic cells and may prove beneficial in future clinical gene transfer or therapy trials.