Biomedical subjects

R Tomanin

Publications and source records attributed to R Tomanin.

At least 19 recordsLinked to original sources

PubMed2004

Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction.

The use of viruses to transduce genes of interest into mammalian cells has been extremely revolutionary, both in terms of laboratory research and for clinical purposes. This approach has allowed expression and over-expression of proteins of interest as well as the understanding of both virus life cycles and eukaryotic cell mechanisms. Beginning in the late eighties gene transduction has been applied to clinical trials but mainly restricted to cancer treatment and genetic diseases. More recently it has been proposed for the cure of infectious diseases (AIDS), vascular diseases and others (Alzheimer's and Parkinson's disease). Viral vectors have been progressively modified in order to increase their transduction efficiency and to reduce their toxicity, immunogenicity and inflammatory potential. In this respect, much has been done in the last few years. By adding genes belonging to other viral species to the vectors' DNA, scientists were able to re-direct their tissue-specificity or to control protein expression. More recently, in the attempt of overcoming the limitations of each viral species, so-called chimeric viral vectors have been generated by combining favourable features of two or more different viruses into one. This review summarises the main characteristics of the most common viral vectors, including their advantages, limitations and possible future applications. It also briefly discusses development and evolution of chimeric vectors, treated in more details along this entire issue. Finally, we evaluate basic safety aspects, mandatory to consider for the clinical application of viral gene transduction.

Adenoviridae↗

PubMed2002

Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a congenital storage disorder resulting from mutations on the iduronate-2-sulfatase (IDS) gene. The disease shows variable clinical phenotypes from severe to mild with progressive neurological dysfunction. The therapeutic options for treatment of MPS II are limited and currently no specific therapies are available; the problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system (CNS). In this work, as a potential treatment for this disease, the transfer of the recombinant IDS enzyme into brain cells has been studied in vitro. Two different approaches to obtain recombinant IDS have been utilized: production of the recombinant enzyme by a transfected human clone (Bosc 23 cells); production of the recombinant enzyme by adenoviral transduction of neuronal (SK-N-BE) or glial (C6) cells. Our data indicate that the transfected as well as the infected cells produce a large amount of the IDS enzyme, which is efficiently endocytosed into neuronal and glial cells through the mannose 6-phosphate (M6P) receptor system. Somatic gene therapy appears therefore to be suitable to correct IDS deficiency in brain cells.

Adenoviridae↗

PubMed2002

Non-viral transfer approaches for the gene therapy of mucopolysaccharidosis type II (Hunter syndrome).

AIMS: Hunter syndrome is a rare X-linked lysosomal storage disorder caused by the deficiency of the housekeeping enzyme iduronate-2-sulphatase (IDS). Deficiency of IDS causes accumulation of undegraded dermatan and heparan-sulphate in various tissues and organs. Approaches have been proposed for the symptomatic therapy of the disease, including bone marrow transplantation and, very recently, enzyme replacement. To date, gene therapy strategies have considered mainly retroviral and adenoviral transduction of the correct cDNA. In this paper, two non-viral somatic gene therapy approaches are proposed: encapsulated heterologous cells and muscle electro-gene transfer (EGT). METHODS: Hunter primary fibroblasts were co-cultured with either cell clones over-expressing the lacking enzyme or with the same incorporated in alginate microcapsules. For EGT, plasmid vector was injected into mouse quadriceps muscle, which was then immediately electro-stimulated. RESULTS: Co-culturing Hunter primary fibroblasts with cells over-expressing IDS resulted in a three- to fourfold increase in fibroblast enzyme activity with respect to control cells. Fibroblast IDS activity was also increased after co-culture with encapsulated cells. EGT was able to transduce genes in mouse muscle, resulting in at least a tenfold increase in IDS activity 1-5 weeks after treatment. CONCLUSION: Although preliminary, results from encapsulated heterologous cell clones and muscle EGT encourage further evaluations for possible application to gene therapy for Hunter syndrome.

Animals↗

PubMed2000

Large-scale feasibility of gene transduction into human CD34+ cell-derived dendritic cells by adenoviral/polycation complex.

With a view to using multiple injections of anti-cancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34+ cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34+ cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-L-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2%, viability =95%) under both small- and large-scale conditions. The addition of poly-L-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a+/AP+ cells = 85 +/- 3% and 80 +/- 2% respectively). Polycations made it possible to reduce the amounts of viral particles, with high efficiency of transduction being achieved at a MOI of 100 with 10 microg/ml poly-L-lysine (CD1a+/AP+: 68 +/- 9%) or 30 microg/ml LipofectAmine (CD1a+/AP+: 60 +/- 7%). Evaluation of the immunophenotype of the transduced DCs showed that the lack of a DC subpopulation was more susceptible to adenoviral transduction. Cryopreservation of transduced DCs did not modify the viability or percentage of AP+ cells that maintain antigen-presenting cell (APC) functions. These findings indicate the efficacy of this method for the transduction of large amounts of CD34+ cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopreservation of transduced DCs did not damage their viability or APC functions, thus making it possible to plan multiple injections of engineered DC-based vaccines.

Adenoviridae↗

PubMed1999

Recombinant adenoviral vector-lipofectAMINE complex for gene transduction into human T lymphocytes.

We have evaluated, as a vector for gene transfer into human T lymphocytes, a recombinant adenovirus (rAd-MFG-AP) carrying a modified, membrane-exposed, alkaline phosphatase (AP) as reporter gene. CD3+ cells were selected from the buffy coat of healthy donors by the immunomagnetic technique. The positive cell population, comprising 96+/-2% CD3+ cells, was cultured with clinical-grade cytokine(s) for 3-7 days prior to rAd-MFG-AP transduction and the transgene expression was evaluated 48 hr later by indirect immunofluorescence flow cytometry assay with an anti-alkaline phosphatase antibody. The best efficiency of transduction was achieved on incubation of CD3+ cells with IL-2 plus either IL-12 (AP+ cells, 12+/-3%) or IL-7 (AP+ cells, 11+/-3%). To increase further the efficiency of transduction, we have combined LipofectAMINE and rAd-MFG-AP with the aim to enhance the uptake of viral particles into the target cells. The percentage of CD3+ cells transduced by rAd-MFG-AP-LipofectAMINE complex was 24+/-4% (range, 20-35%) after incubation with IL-2 plus IL-7 and 22+/-4% (range, 18-32%) after incubation with II-2 plus IL-12. Forty-eight hours after the incubation with rAd-MFG-AP, the transduced T lymphocytes were subjected to fluorescence-activated cell sorting and fractionated into AP+ and AP- cell subpopulations. The AP+ cell fraction, comprising 96.8% of AP+ cells, was evaluated by FACScan analysis for T lymphocyte surface antigens. The immunophenotyping of the transduced T lymphocytes has shown that there was not a particular subtype of T lymphocytes more susceptible to rAd-MFG-AP transduction. In addition, the transgene expression did not modify T lymphocyte functions, as demonstrated by results obtained by cytotoxicity assay before and after rAd-MFG-AP-LipofectAMINE complex transduction. In conclusion, human T lymphocytes can be efficiently transduced, under clinically applicable conditions, by adenovirus-LipofectAMINE complex after 7 days of culture with IL-2 and IL-12 or IL-7.

Adenoviridae↗

PubMed1997

The bacteriophage T7 binary system activates transient transgene expression in zebrafish (Danio rerio) embryos.

The bacteriophage T7 binary expression system is widely used in vitro for high level selective expression of cloned genes but its application to in vivo models has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing the T7 RNA polymerase gene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing the Escherichia coli lacZ gene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary system in vivo to a vertebrate model.

Animals↗

PubMed1997

Development and characterization of a binary gene expression system based on bacteriophage T7 components in adenovirus vectors.

To explore the utility of the bacteriophage T7 binary system in adenovirus (Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase produced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial beta-galactosidase (betaGal) (lacZ) gene under the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7betaGal). Coinfections were performed with the various AdT7pol vectors and the reporter vector, and expression was analysed in three different human cell lines: 293, A549 and MRC-5. Depending on the AdT7pol vector used, different levels of expression were obtained from the reporter gene. In 293 cells, expression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7betaGal. In A549 and MRC-5 cells very little expression was detected using AdT7pol1 or pol2 and efficient expression was only obtained when relatively high moi values of the replication-competent vector were used in the coinfections. We also constructed a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evaluate the 'leakiness' of the Ad-T7 system detected very little expression from the T7pro in the absence of T7 polymerase suggesting this system may be useful for the cloning and expression of genes encoding cytotoxic proteins.

Adenoviridae↗

PubMed1997

In vitro correction of iduronate-2-sulfatase deficiency by adenovirus-mediated gene transfer.

Hunter syndrome is a lethal lysosomal storage disorder caused by the deficiency of iduronate-2-sulfatase and characterized by severe skeletal and neurological symptoms. Only symptomatic treatments are available and, although bone marrow transplantation has been suggested, no encouraging results have been obtained so far. Therefore, gene therapy might be a route to be pursued for treatment of the disease. In this respect, one major goal to achieve is the generation of an overexpressing vector able to correct, in particular, central nervous system (CNS) cells. Adenoviruses have been shown to infect CNS cells efficiently with minor or even absent immunological response. We describe the generation of a replication-defective adenoviral vector, AdRSVIDS, which is able to express in vitro high levels of iduronate-2-sulfatase. After infection, accumulation of mucopolysaccharides in treated Hunter cells was normalized. Furthermore, endocytosis of the transduced IDS did occur via the mannose-6-phosphate (M6P) receptor. Since no animal model for the disease is available, we developed a system based on the generation of derma-equivalents which enabled us to verify the expression of high levels of sulfatase up to 30 days after infection.

Adenoviridae↗

PubMed1994

Antiretroviral activity of furocoumarins plus UVA light detected by a replication-defective retrovirus.

The replication defective retrovirus, pXM5(N2), was used for an easy, safe and reproducible test for the screening of furocoumarins with antiretroviral activity. High titer viral supernatants have been photomodified by UVA light (20 kJ m-2) in the presence of different concentrations of two psolarens (8-methoxypsoralen, 8-MOP and 4,5',8-trimethylpsoralen, TMP) and one angelicin (4,6,4'-trimethylangelicin, TMA). At low concentrations (100-250 ng ml-1) 8-MOP and TMA did not show any significant antiviral activity, while TMP demonstrated a reduction of virus infectivity by one log at 250 ng ml-1. At the highest concentration (5 micrograms ml-1), TMA and TMP reduced the virus titer by one and more than two logs, respectively, being, therefore, two and four times more active than 8-MOP. The most active compound, TMP, was further tested on HIV-1 viral supernatants. Total inactivation of the HIV-1 (200 SFU) was obtained in the presence of 1 microgram ml-1 of TMP and 20 kJ m-2 of UVA light. Our results support the validity of the N2 system to detect the antiretroviral activity of furocoumarins and suggest the potential of TMP in combination with UVA light against HIV-1.

3T3 Cells↗

PubMed1993

Generation of a new adenovirus type 12-inducible fragile site by insertion of an artificial U2 locus in the human genome.

Infection with adenovirus type 12 (Ad12) induces four fragile sites in the human genome (H.F. Stich, G.L. van Hoosier, and J.J. Trentin, Exp. Cell Res. 34:400-403, 1964; H. zur Hausen, J. Virol. 1:1174-1185, 1967). The major site, at 17q21-22, contains the U2 gene cluster, which is specifically disrupted by infection in at least a percentage of the cells (D.M. Durnam, J.C. Menninger, S.H. Chandler, P.P. Smith, and J.K. McDougall, Mol. Cell. Biol. 8:1863-1867, 1988). For direct assessment of whether the U2 locus is the target of the Ad12 effect, an artificial locus, constructed in vitro and consisting of tandem arrays of the U2 6-kbp monomer, was transfected into human cells. We report that integration of this artificial locus on the p arm of chromosome 13 creates a new Ad12-inducible fragile site.

Adenoviruses, Human↗

PubMed1992

Chromosome aberrations and micronuclei in lymphocytes of workers exposed to low and medium levels of styrene.

In the present study we analysed 19 workers exposed to styrene in two factories where polyester resins were used. Because of the different sizes of the pieces undergoing resin processing, the environmental styrene concentrations and urinary mandelic acid (MA) levels of the analysed subjects were quite different in the two plants examined. Cytogenetic monitoring was performed by analysis of chromosome aberrations (CAs) and micronuclei (Mn) in peripheral blood lymphocytes. Cytogenetic analysis revealed a significant increase in the percentage of aberrant cells and total aberrations in the group with higher styrene exposure (group 2) and no increase in the group with lower exposure (group 1), as compared with matched controls. Mn frequencies were not significantly increased in the two exposed populations. No correlations between length of exposure and CA or Mn frequency were found, and a weak correlation was found between exposure levels, measured as urinary MA, and Mn frequencies. Only 5 of the 12 exposed workers examined in group 2 had urinary MA levels higher than the limit recommended by the ACGIH in 1990-91 [1]. Significant increases in DNA damage are therefore already found at urinary MA levels lower than the internationally suggested exposure limits.

Adult↗

PubMed1991

Influence of smoking habit on the frequency of micronuclei in human lymphocytes by the cytokinesis block method.

To evaluate the frequency of micronucleated lymphocytes in a normal population, 45 healthy subjects were analysed by using a modified cytochalasin B block method; the influence of some confounding factors (sex, age, smoking, etc.) were taken into account. Using a stepwise regression test smoking habits were found to have a statistically significant influence on the frequency of micronucleated cells and micronuclei. In addition, the mitotic and proliferative indexes, and the frequency of micronucleated lymphocytes, at different culture times, were studied in four healthy subjects. Based on the results we suggest that cytochalasin B be added to cultures no later than the 42nd hour.

Adult↗

PubMed1991

Studies of biological and chemical monitoring of low-level exposure to ethylene oxide.

Ten sanitary workers exposed to concentrations of ethylene oxide below 1 ppm were studied to determine whether effects could be observed at low exposure levels. A significant increase in the number of sister chromatid exchanges in cultured lymphocytes was found only for five subjects with relatively high exposure in the sterilization area. However, it was not possible to separate clearly the effect of smoking from that of ethylene oxide exposure. No increase in the frequencies of micronuclei in lymphocytes and buccal cells was found. The level of 2-hydroxyethyl adducts to the N-terminal valines in hemoglobin responded in a reliable fashion to chronic ethylene oxide exposure and smoking. Furthermore, measurement of levels of 2-hydroxyethyl adducts to the N-terminal valines in hemoglobin made it possible to reconstruct the dynamics of a leakage of ethylene oxide which involved three workers.

Adult↗

PubMed1990

Chromosomal aberrations, sister chromatid exchanges, and urinary thioethers in nurses handling antineoplastic drugs.

In this study we examined the structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) of 12 nurses handling moderate quantities of antineoplastic drugs. Urinary thioethers were also measured to assess the potential exposure to alkylating drugs. Two control groups with similar mean age and smoking habits and working in the same hospital were also studied: 1) control clerks and 2) control nurses. Our study did not show any clear increase of chromosomal damage in exposed nurses as compared to controls. However, CA in control nurses were significantly increased (p = 0.05) with respect to control clerks. The results of baseline urinary excretion of thioethers were statistically higher (p less than 0.02) in exposed subjects than in control clerks.

Antineoplastic Agents↗

PubMed1990

Louis-Bar syndrome: spontaneous and induced chromosomal aberrations in lymphocytes and micronuclei in lymphocytes, oral mucosa and hair root cells.

Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.

Adolescent↗

PubMed1990

Evaluation of chromosomal aberrations in lymphocytes and micronuclei in lymphocytes, oral mucosa and hair root cells of patients under antiblastic therapy.

In 7 patients undergoing antiblastic chemotherapy for the first time, the structural chromosomal aberration (CA) test in peripheral lymphocytes was compared with the micronucleus (Mn) test in lymphocytes, in oral cavity cells and in hair root cells of the scalp. The last test is being proposed for the first time. The CA and Mn frequencies induced by chemotherapy were compared with the baseline (pretreatment) frequencies of the patients and with confidence limits calculated in 4 control groups studied for CA, Mn in lymphocytes, Mn in oral cavity cells and Mn in hair root cells, respectively. The studied chemotherapies induced a clear cytogenetic effect in at least 2 of the tests studied with the exception of interferon-alpha 2b (patient 6) and interferon + low doses of cis-platinum (patient 2) which did not appear to cause evident chromosomal damage. The response to chemotherapy is generally characterized by an increase in Ca and Mn, reaching a peak value and then decreasing in the following weeks. The CA test proves to be the most sensitive despite the fact that CA were analyzed in an average of 100 cells per sample against the 500-3000 cells analyzed for Mn. The efficiency of Mn to detect CA is in the following order: Mn in lymphocytes greater than Mn in buccal cells greater than Mn in hair root cells. The last test appears to be very promising but, used following the current method, does not appear suitable to monitor acute exposure.

Adult↗

PubMed1990

The micronucleus assay in human exfoliated cells of the nose and mouth: application to occupational exposures to chromic acid and ethylene oxide.

We have applied the micronucleus (MN) assay to exfoliated cells of buccal and nasal cavities to monitor the genotoxic risk in a group of workers exposed to chromic acid and in another group exposed to ethylene oxide (EtO). The first group comprised 16 subjects working in a 'hard' type chrome-plating factory showing increased chromium absorption and chromium-induced rhinopathy. The second group comprised 9 subjects working in a sterilization unit, exposed to EtO concentrations lower than 0.38 ppm as timed weighted average (TWA) for a working shift; 3 of them were involved in a acute exposure too. The frequency of MN in buccal mucosa was within the norm for exposure both to chromium and to EtO. The MN frequency in nasal mucosa was not altered in chromium platers, whereas a significant increase (p less than 0.01) in MN was found in 2 out of 3 subjects involved in the accidental EtO leakage and a non-significant increase in MN was found in the group chronically exposed to EtO.

Adult↗

PubMed1989

Chromosomal alterations in peripheral blood lymphocytes, urinary mutagenicity and excretion of polycyclic aromatic hydrocarbons in six psoriatic patients undergoing coal tar therapy.

Six male non-smoking subjects treated for psoriasis with topical applications of pure coal tar or 4% coal tar-containing ointment were examined in order to assess the genotoxic risk associated with this type of therapy. Mutagenicity in urine samples collected before and during the coal tar therapy was evaluated in the plate incorporation assay on Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. Total urinary polycyclic aromatic hydrocarbon (PAH) levels were evaluated in parallel by high resolution gas chromatography/mass spectrometry. In addition, sister chromatid exchanges and chromosomal aberrations were also analysed in peripheral blood lymphocytes collected before, during and after the end of the coal tar applications. The results suggest that urinary mutagenicity levels as well as the frequencies of chromosome aberrations and sister chromatid exchanges in lymphocytes are related to the levels of exposure to coal tar. Moreover the kinetics of repair of chromosome damage in relation to different exposure levels and the capacity of the urinary mutagenicity assay to correctly identify the exposure to significant levels of PAH are discussed.

Administration, Topical↗