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D Valerio

Publications and source records attributed to D Valerio.

At least 73 records · Page 4Linked to original sources
PubMed1995

Aerosol-mediated delivery of recombinant adenovirus to the airways of nonhuman primates.

At present, it is conceivable that gene therapy of the cystic fibrosis airway epithelium is possible using the direct transfer of a functional human cystic fibrosis transmembrane conductance regulator (CFTR) gene to a wide variety of patients' tracheo-bronchial cells. Here we describe a novel approach (aerosolization) to deliver a replication-deficient adenovirus carrying the CFTR gene (Ad.CFTR) to the airways. Results obtained in vitro and in Rhesus monkeys suggest that the delivery of recombinant adenovirus as an aerosol is feasible and is not associated with severe toxicity after single or double administration depending on the Ad.CFTR dose. This study supports the concept of aerosolization as a delivery method for adenovirus-mediated lung gene therapy.

Adenoviridae↗
PubMed1995

Trans-complementation of E1-deleted adenovirus: a new vector to reduce the possibility of codissemination of wild-type and recombinant adenoviruses.

Treatment of cystic fibrosis by gene therapy will require the development of vectors capable of efficient and safe transfer of a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. To achieve this goal, replication-deficient (E1-) adenoviruses (Ad) are promising vectors. We have previously demonstrated efficient CFTR gene delivery to the airways of cotton rats and rhesus monkeys using a replication-deficient adenovirus, Ad-CFTR. Here, we have investigated an important safety issue, the interaction between the vector and wild-type virus which can provide the missing E1 function in trans. We show that Ad5 can mobilize the defective Ad-CFTR genome in vitro and in cotton rats. However, the extent of the complementation in vivo by wild-type virus is limited because no additional spreading or shedding of Ad-CFTR to trachea, lungs, and stools is elicited. To attenuate Ad-CFTR further, a mutation was introduced in the cis-acting regulatory sequences that control the encapsidation of the viral genome. We demonstrate that when cells are coinfected with wild-type virus and the new attenuated vector, the viral DNA containing the natural encapsidation sequences is preferentially packaged, leading to a rapid dilution of the recombinant virus.

Adenoviridae↗
PubMed1995

Gene therapy for adenosine deaminase deficiency.

In the last decade, gene transfer into hematopoietic cells has evolved from an experimental procedure which resulted in successfully transduced in vitro hematopoietic colonies to the first clinical trials in patients suffering from severe combined immunodeficiency disease caused by the absence of functional adenosine deaminase. Significant in vivo expression of the newly introduced gene encoding human adenosine deaminase has been observed in descendents of murine and rhesus monkey hematopoietic stem cells following retrovirus mediated gene transfer. So far, 10 patients have received genetically repaired T-cells, hematopoietic stem cells or both without the appearance of any side effect. The clinical bone marrow gene transfer studies differ largely from the monkey studies with respect to myeloablation, which was applied in the monkey studies, but not in the patient studies. Ongoing studies in patients show that the introduced gene is present in circulating blood cells. In the initial phase of the trial, the frequency of transduced circulating blood cells is lower than in rhesus monkey studies. This difference may be contributed to the fact that conditioning was not performed in the patients.

Adenosine Deaminase↗
PubMed1995

Expression and localization of CFTR in the rhesus monkey surface airway epithelium.

The Rhesus monkey has been used as a model for evaluating the possibility of introducing the CFTR gene into the airway epithelium in vivo. We addressed the question of whether the simian airway surface epithelium exhibits a CFTR distribution and functional activity (ciliary beating frequency) similar to that of human airway surface epithelium. Expression of CFTR mRNA was demonstrated on Rhesus monkey tracheobronchial tissue by reverse transcription polymerase chain reaction (RT-PCR) analysis. By immunofluorescent light microscopy, CFTR was localized on the apical plasma membrane of ciliated cells as we previously described for human tracheobronchial surface epithelium. The ciliary beat frequency (CBF) measured on the explant of Rhesus monkey tracheobronchial tissue appeared to be similar to that of the CBF of human tracheobronchial ciliated cells. To compare the Rhesus monkey CFTR gene with that of the human, we sequenced parts of exon 13 (encoding the R domain) and exon 24 (encoding the C terminal part of the protein) of the Rhesus monkey CFTR gene. The nucleotide sequence identity with the human counterpart was found to be 98% and 94% respectively, although restriction enzyme differences allow discrimination between Rhesus monkey and human CFTR cDNA. Taken together, these results suggest that the airway epithelium of the Rhesus monkey is a suitable model of human respiratory epithelium for analyzing the effect of human CFTR gene transfer.

Amino Acid Sequence↗
PubMed1995

Influence of interleukin-3, interleukin-6, and stem cell factor on retroviral transduction of rhesus monkey CD34+ hematopoietic progenitor cells measured in vitro and in vivo.

As a preclinical test for bone marrow gene therapy, we transduced Rhesus monkey CD34+CD11b- hematopoietic progenitor cells with recombinant retroviruses. We investigated the effects of the recombinant hematopoietic growth factors interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) on the susceptibility of in vitro clonogenic progenitor cells and in vivo repopulating stem cells to retroviral transduction. IL-6 did not contribute to transduction of progenitor cells, whereas IL-3 and SCF supported expansion and transduction of progenitors. The combination of IL-3 and IL-6 was most efficient at promoting transduction of more mature progenitor cell types. Cultures containing IL-6+SCF yielded optimal maintenance of CD34+CD11b- cells without evidence for lineage-restricted maturation. Autologous transplantation of transduced grafts cultured in the presence of SCF, with or without IL-3 or IL-6, into lethally irradiated Rhesus monkeys resulted in a severely delayed hematopoietic reconstitution as compared with grafts transduced in the presence of IL-3 alone. After in vivo repopulation, transduced cells were found among peripheral blood mononuclear cells, granulocytes and CD34+CD11b- progenitor cells in the bone marrow of engrafted animals. However, no significant difference in transduction efficiency on in vivo repopulating stem cells could be demonstrated among the tested growth factor conditions.

Animals↗
PubMed1995

Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer.

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.

Animals↗
PubMed1994

Proliferative response of hepatocytes transplanted into spleen or solid support.

Understanding the regenerative behavior of transplanted hepatocytes is of great importance for developing and improving such novel therapeutic strategies as hepatocellular transplantation and ex vivo gene therapy. In this study the proliferative responsiveness of transplanted syngeneic rat hepatocytes was examined in relation to the timing of the administration of a mitogenic stimulus. For this purpose nuclear bromodeoxyuridine incorporation after partial hepatectomy was investigated during the early post-transplant phase. The response of intrasplenically transplanted hepatocytes was compared to that of liver cells engrafted in polytetrafluoroethylene solid supports that had been implanted intraperitoneally 4 weeks prior to transplantation. Nonstimulated, engrafted hepatocytes exhibited a labeling index of approximately 0-1% independent of the transplantation technique used. This "spontaneous" labeling index did not change with time. Partial hepatectomy, executed simultaneously with hepatocyte transplantation through either technique, did not result in significant alteration of this proliferation index. Delayed kinetics were found not to be responsible for this lack of responsiveness. When the mitogenic stimulus was given between 2 and 6 weeks post-transplantation, a significant increase in labeling index was observed in comparison to sham-treated control animals. Maximal labeling indices of approximately 3-4% were found if the stimulus took place at 4 weeks post-transplantation. Both the pattern and the extent of the proliferative response seen in liver cells engrafted in solid supports were similar to the ones found in intrasplenic hepatocytes, indicating adequate vascularization of the supports. This data provides the first description of proliferative response in hepatocytes transplanted by the solid support technique, which may offer an attractive alternative to the intrasplenic route.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Lung gene therapy: in vivo adenovirus-mediated gene transfer to rhesus monkey airway epithelium.

Somatic gene therapy of lung disorders such as cystic fibrosis (CF) aims at introducing the therapeutic gene into respiratory epithelium. We have tested the ability of recombinant human adenovirus to infect rhesus monkey airway epithelium in vivo. Application of adenovirus harboring the lacZ marker gene to the airway surface resulted in large patches of lacZ-positive cells in the trachea, bronchi, and bronchioles, 6 days after virus exposure, indicating a successful transfer of the lacZ gene to respiratory epithelium. Microscopic analysis showed that basal, mucous goblet, and ciliated cells were lacZ positive. In addition, gene transfer to the submucosal glands was observed. Pathological examination of the organs revealed no virus-mediated toxic effects to the lungs and other organs. Using polymerase chain reaction (PCR) analysis we found no spread of the virus to blood or any organ tested. These results indicate the potential use and safety of adenoviruses as a tool in human gene therapy procedures aimed at pulmonary diseases.

Adenoviruses, Human↗
PubMed1994

Gene transfer into nonhuman primate CD34+CD11b- bone marrow progenitor cells capable of repopulating lymphoid and myeloid lineages.

We investigated whether rhesus monkey CD34+CD11b- hematopoietic stem cells can be transduced with recombinant retroviruses carrying the human adenosine deaminase (hADA) gene by co-cultivation with a virus-producing cell line. Following autologous transplantation, polymerase chain reaction (PCR) analysis on peripheral blood mononuclear cells and granulocytes showed that the hADA-retrovirus was present in approximately 0.1% of the cells for at least 400 days post transplantation in 2 monkeys. Bone marrow that was harvested 16 months after transplantation carried ADA-overexpressing myeloid progenitor cells capable of in vitro colony formation. In addition, hADA activity could be demonstrated in T lymphocytes that were harvested 9 months post transplantation. Thus, in vitro transduction of CD34+CD11b- cells led to long-term repopulation of the hematopoietic system with transduced cells of lymphoid and myeloid lineages expressing the hADA gene. To investigate whether infusion of virus-producing cells into a rhesus monkey undergoing autologous bone marrow transplantation could lead to in vivo transfer of the recombinant retrovirus, 1 monkey was infused with CD34+CD11b- bone marrow cells (BMC) and a large quantity of virus-producing cells. Few provirus-carrying cells could temporarily be detected in this animal. This shows that in vivo gene transfer into a regenerating hemopoietic system can occur, albeit at very low efficiency.

Adenosine Deaminase↗
PubMed1994

In vivo adenovirus-mediated transfer of human CFTR cDNA to rhesus monkey airway epithelium: efficacy, toxicity and safety.

We have administered replication defective recombinant adenovirus harboring the human CFTR cDNA (Ad.CFTR) to lungs of Rhesus monkeys and assessed toxicity, efficiency of gene transfer and containment of recombinant adenovirus in the lungs. Gene transfer efficiencies, as measured by PCR analysis, were dose-dependent. Administration of low dose Ad.CFTR [1.5 x 10(7) plaque forming units (p.f.u.)] was not accompanied by any clinical chemical or histopathological changes. Mild to moderate multifocal perivascular and peribronchial lymphocytic infiltrates were found upon histopathological analysis only after administration of a high dose of recombinant adenovirus, although not accompanied by changes in clinical chemical parameters. Long-term expression (up to 128 days) was found after administration of recombinant adenovirus. Re-challenging of the monkeys treated with high-dose recombinant adenovirus resulted again in gene transfer at all levels of lungs and airways, without being accompanied by additional histopathological changes. Circulating anti-adenovirus antibodies were elicited. Animals treated with high-dose adenovirus secreted virus in pharynx and faeces for maximally 2 and 4 days after administration, respectively. These results show that recombinant adenovirus can be used for efficient delivery of genes into primate airway epithelium without signs of severe toxicity.

Adenoviridae↗
PubMed1993

Factors affecting the transduction of pluripotent hematopoietic stem cells: long-term expression of a human adenosine deaminase gene in mice.

An amphotropic retroviral vector, LgAL(delta Mo + PyF101) containing a human adenosine deaminase (ADA) cDNA was used to optimize procedures for the lasting genetic modification of the hematopoietic system of mice. The highest number of retrovirally infected cells in the hematopoietic tissues of long-term reconstituted mice was observed after transplantation of bone marrow (BM) cells that had been cocultured in the presence of both interleukin-1 alpha (IL-1 alpha) and IL-3. A significantly lower number was detected when IL-1 alpha was omitted from such cocultures. The yield of cells that generate spleen colony-forming cells (CFU-S) in the BM of lethally irradiated recipients (MRA-CFU-S) significantly improved on inclusion of the adherent cell fraction of cocultures in the transplant. Retroviral integration patterns in MRA-CFU-S-derived spleen colonies showed that an MRA-CFU-S can produce many CFU-S during BM regeneration. Expression of hADA was detected in the circulating white blood cells of long-term reconstituted animals, demonstrating that the LgAL(delta Mo + PyF101) vector is capable of directing the sustained expression of hADA, and in approximately 35% of the transduced MRA-CFU-S-derived spleen colonies. These results should facilitate the development of gene therapy protocols for the treatment of severe combined immunodeficiency caused by a lack of functional ADA.

Adenosine Deaminase↗
PubMed1993

Circumvention of chemotherapy-induced myelosuppression by transfer of the mdr1 gene.

Drug-induced myelosuppression is a frequent reason for curtailing chemotherapy in cancer patients. 'Rescue' of myelosuppressed patients with autologous marrow transplants is reasonably advanced and permits an increase in the dose of anticancer drugs. Despite this improvement, patients often relapse with drug resistance disease. The human multidrug resistance (mdr1) gene might make it possible to render hemopoietic stem cells resistant to anticancer drugs after transfer of this gene. By introducing resistant stem cells into patients it might be possible to treat these patients repeatedly with otherwise ablative therapy. This review explores the feasibility of mdr1 gene therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Retrovirus-mediated gene transfer into rhesus monkey hematopoietic stem cells: the effect of viral titers on transduction efficiency.

We have generated a cell line, designated POAM-P1, shedding amphotropic recombinant retroviruses carrying the human adenosine deaminase (hADA) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cells and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduction efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultivation of rhesus monkey bone marrow with POAM-P1 cells, successful transduction could be demonstrated in approximately 10% of myeloid progenitor colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PBMC) and granulocytes in vivo until > 1 year after autologous transplantation. In addition, the presence of functional hADA enzyme was detected in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for > 2 years after transplantation. Despite the higher retrovirus titer of POAM-P1 cells as compared to POC-1 cells, no difference was observed in gene transfer efficiency into CFU-C and long-term repopulating stem cells. This shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic stem cells.

3T3 Cells↗
PubMed1993

A new case of partial trisomy 19q (q13.2-->qter) owing to an unusual maternal translocation.

A new case of trisomy 19q13.2-->qter is described in a male child which was caused by a maternal balanced translocation (13;19)(p13;q13.2). The major clinical features detected in the patient included the following: facial dysmorphism, bilateral coloboma, narrow and hypoplastic nails, cardiac malformations (Fallot's tetralogy), genitourinary and gastrointestinal anomalies, and agenesis of the corpus callosum. A comparison with other reported cases of partial trisomy 19q is presented. A hypothesis is proposed to account for the involvement of p13 regions of different acrocentrics in some cases of familial translocations involving a chromosome 19.

Chromosomes, Human, Pair 19↗
PubMed1993

Bone marrow gene therapy for adenosine deaminase deficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency disease (SCID). The cause for this is believed to be the accumulation of one of the substrates for ADA, 2'-deoxyadenosine to which especially T cells are hypersensitive. This disease can be treated successfully with bone marrow transplantation if a suitable donor is available. Alternatively, the human ADA gene could be introduced into the autologous bone marrow. We have generated a retroviral vector containing the human ADA gene. With this vector we were able to restore human ADA-activity in ADA-SCID T cells to normal levels resulting in a sensitivity to 2'-deoxyadenosine that is also found for T cells from a healthy donor. In murine studies we have shown that our retrovirus can infect pluripotent hemopoietic stem cells resulting in long-term (> 6 months) expression of human ADA in the hemopoietic system of transplanted animals. These results were confirmed in rhesus monkeys where we were able to detect the provirus in both peripheral blood mononuclear cells and granulocytes for as long as the animals were analyzed, i.e. up to more than 1 year post bone marrow transplantation. On the basis of these results we have proposed a clinical protocol for the treatment of ADA-SCID patients with bone marrow gene therapy.

Adenosine Deaminase↗