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Biomedical subjects

V Mautner

Publications and source records attributed to V Mautner.

At least 19 recordsLinked to original sources
PubMed2006

Use of synthetic vectors for neutralising antibody resistant delivery of replicating adenovirus DNA.

Use of synthetic vectors to deliver genomes of conditionally replicating lytic viruses combines the strengths of viral and non-viral approaches by enabling neutralising antibody resistant deployment of cancer virotherapy. Adenovirus is particularly suitable for this application since all proteins essential for replication can be expressed from the input DNA, although the presence of terminal protein (TP) covalently linked to the 5' termini of the input virus genomes both improves expression of transgenes encoded in the input DNA and also enhances replication. These roles of TP were distinguished in experiments where E1-deleted Ad(GFP)DNA bearing TP (Ad(GFP)DNA-TP), delivered with DOTAP, gave a two-fold greater frequency of transduction than Ad(GFP)DNA(without TP) in non-complementing A549 cells, while in 293 cells (which support replication of E1-deleted viruses) the presence of TP mediated a much greater differential transgene expression, commensurate with its ability to promote replication. Subsequent studies using AdDNA for virotherapy, therefore, included covalently linked TP. AdDNA-TP delivered to A549 cells using a synthetic polyplex vector was shown to be resistant to levels of neutralising antisera that completely ablated infection by wild-type adenovirus, enabling polyplex/Ad(wild type)DNA-TP to mediate a powerful cytopathic effect. Similarly in vivo, direct injection of a polyplex/Ad(wild type)DNA-TP into A549 tumours was neutralising antibody-resistant and enabled virus replication, whereas intact virus was neutralised by the antibody and failed to infect. The delivery of adenovirus genomes-TP using synthetic vectors should provide a strategy to bypass neutralising antibodies and facilitate clinical application of replicating adenovirus for cancer virotherapy.

Adenoviridae↗
PubMed2006

Chick embryo lethal orphan virus can be polymer-coated and retargeted to infect mammalian cells.

Non-human adenovirus vectors have attractive immunological properties for gene therapy but are frequently restricted by inefficient transduction of human target cells. Using chicken embryo lethal orphan (CELO) virus, we employed a nongenetic mechanism of polymer coating and retargeting with basic fibroblast growth factor (bFGF-pc-CELOluc), a strategy that permits efficient tropism modification of human adenovirus. bFGF-pc-CELOluc showed efficient uptake and transgene expression in chick embryo fibroblasts (CEF), and increased levels of binding and internalization in a variety of human cell lines. Transgene expression was also greater than unmodified CELOluc in PC-3 human prostate cells, although the specific activity (RLU per internalized viral genome) was decreased. In CEF, the specific activity of bFGF-pc-CELOluc was considerably higher than in the human prostate cell line PC-3. Retargeted virus was fully resistant to inhibition by human serum with known adenovirus-neutralizing activity in vitro, while in mice CELOluc was cleared less rapidly from the blood than Adluc following i.v. administration in the presence of adenovirus neutralizing serum. Polymer coating and retargeting with bFGF further reduced rates of clearance for both viruses, suggesting protection against both neutralizing and opsonizing factors. The data indicate that CELO virus may be retargeted to infect human cells via alternative, potentially disease-specific, receptors and resist the effects of pre-existing humoral immunity.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2005

Identification of novel deletion breakpoints bordered by segmental duplications in the NF1 locus using high resolution array-CGH.

BACKGROUND: Segmental duplications flanking the neurofibromatosis type 1 (NF1) gene locus on 17q11 mediate most gene deletions in NF1 patients. However, the large size of the gene and the complexity of the locus architecture pose difficulties in deletion analysis. We report the construction and application of the first NF1 locus specific microarray, covering 2.24 Mb of 17q11, using a non-redundant approach for array design. The average resolution of analysis for the array is approximately 12 kb per measurement point with an increased average resolution of 6.4 kb for the NF1 gene. METHODS: We performed a comprehensive array-CGH analysis of 161 NF1 derived samples and identified heterozygous deletions of various sizes in 39 cases. The typical deletion was identified in 26 cases, whereas 13 samples showed atypical deletion profiles. RESULTS: The size of the atypical deletions, contained within the segment covered by the array, ranged from 6 kb to 1.6 Mb and their breakpoints could be accurately determined. Moreover, 10 atypical deletions were observed to share a common breakpoint either on the proximal or distal end of the deletion. The deletions identified by array-CGH were independently confirmed using multiplex ligation-dependent probe amplification. Bioinformatic analysis of the entire locus identified 33 segmental duplications. CONCLUSIONS: We show that at least one of these segmental duplications, which borders the proximal breakpoint located within the NF1 intron 1 in five atypical deletions, might represent a novel hot spot for deletions. Our array constitutes a novel and reliable tool offering significantly improved diagnostics for this common disorder.

Chromosome Breakage↗
PubMed2004

Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector.

Viruses that replicate selectively in cancer cells constitute an exciting new class of anticancer agent. The conditionally replicating adenovirus (CRAd) dl1520, which lacks the E1B-55K gene, has elicited significant clinical responses in humans when used in combination with chemotherapy. A convergent development has been to use replication-defective viruses to express prodrug-activating enzymes in cancer cells. This can sensitize the cancer to prodrug, but depends upon achieving sufficient level, distribution and specificity of enzyme expression within the tumour. In this study, we have expressed the prodrug-activating enzyme nitroreductase (NTR) in the context of an E1B-55K-deleted adenovirus, CRAd-NTR(PS1217H6). We show that CRAd-NTR(PS1217H6) retains oncolytic growth properties, and expresses substantially more NTR than a comparable, replication-defective adenovirus. The combination of viral oncolysis and NTR expression results in significantly greater sensitization of SW480 and WiDr colorectal cancer cells to the prodrug CB1954 in vitro. In vivo, CRAd-NTR(PS1217H6) was shown to replicate in subcutaneous SW480 tumour xenografts in immunodeficient mice, resulting in more NTR expression and greater sensitization to CB1954 than with replication-defective virus. Combination therapy of CRAd-NTR(PS1217H6) with CB1954 reduced tumour growth from 13.5- to 2.8-fold over 5 weeks, and extended median survival from 42 to 81 days, compared with no treatment.

Adenoviridae↗
PubMed2004

Extended plasma circulation time and decreased toxicity of polymer-coated adenovirus.

Systemic delivery of adenoviral vectors is a major goal in cancer gene therapy, but is currently prohibited by rapid hepatic uptake of virus following intravenous injection with levels of viable virus in the murine plasma typically falling to less than 0.1% after 30 min. We have used a surface-masking technique based on multivalent copolymers of poly(N-(2-hydroxypropyl)methacrylamide) to ablate all pathways of receptor-mediated infection, combined with dose modulation to achieve partial saturation of nonspecific uptake pathways. Polymer coating gave at least 100-fold decreased hepatic transgene expression at all doses and even high doses of coated virus (pc-virus) showed no weight loss or stimulation of serum transaminases. Low doses of virus and pc-virus (10(9) viral particles (vp)/mouse) were mainly captured by the liver (assessed by quantitative PCR), although higher doses led to greater fractional persistence in the plasma (measured after 30 min). Coated virus at a dose of 6 x 10(11) vp/mouse showed nearly 50% plasma circulation, representing a 3.5-fold greater area under the concentration-time curve (0-30 min) compared to unmodified virus. Such an increase in the bioavailability of adenovirus, coupled with substantial decreases in toxicity and unwanted transgene expression is an important step towards producing systemically available tumour-targeted viruses.

Adenoviridae↗
PubMed2003

Elevated risk for MPNST in NF1 microdeletion patients.

An NF1 microdeletion is the single most commonly reported mutation in individuals with neurofibromatosis type 1 (NF1). Individuals with an NF1 microdeletion have, as a group, more neurofibromas at a younger age than the group of all individuals with NF1. We report that NF1 microdeletion individuals additionally have a substantially higher lifetime risk for the development of malignant peripheral nerve sheath tumors than individuals with NF1 who do not have an NF1 microdeletion. This should be taken into account in the medical follow-up of individuals with an NF1 microdeletion.

Adolescent↗
PubMed2003

Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas.

Neurofibromatosis 2 (NF2) is a severe autosomal dominant disorder that predisposes to multiple tumours of the nervous system. About half of all patients are founders with clinically unaffected parents. The purpose of the present study was to examine the extent to which mosaicism is present in NF2 founders. A total of 233 NF2 founders with bilateral vestibular schwannomas (BVS) were screened by exon scanning. NF2 mutations were detected in the blood samples of 122 patients (52%). In 10 of the 122 cases, the ratio of mutant to normal alleles was obviously less than 1, suggesting mosaicism. Tumour specimens were available from 35 of the 111 subjects in whom no mutation could be detected in blood specimens. Mutational analysis by exon scanning detected typical NF2 mutations in 21 of the 35 tumours. In nine subjects, the alterations found in tumours could be confirmed to be the constitutional mutation based on finding of identical mutations in pathologically and/or anatomically distinct second tumours. In six other subjects with only a single tumour available, allelic loss of the NF2 gene was found in addition to the mutation in each tumour, suggesting that either the mutation or the deletion of the NF2 gene is probably the constitutional genetic alteration. Our results suggest that failure to find constitutional mutations in blood specimen from these 15 patients was not because of the limitation of the applied screening technique, but the lack of the mutations in their leucocytes, best explained by mosaicism. Extrapolating the rate (15/35 = 43%) of mosaicism in these 35 cases to the 111 NF2 founders with no constitutional NF2 mutations found in their blood, we inferred 48 mosaic subjects (111 x 0.429). Adding the 10 mosaic cases detected directly in blood specimens, we estimate the rate of mosaicism to be 24.8% (58/233) in our cohort of 233 NF2 founders with bilateral vestibular schwannomas.

Base Sequence↗
PubMed2001

High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder whose hallmark is bilateral vestibular schwannoma. It displays a pronounced clinical heterogeneity with mild to severe forms. The NF2 tumor suppressor (merlin/schwannomin) has been cloned and extensively analyzed for mutations in patients with different clinical variants of the disease. Correlation between the type of the NF2 gene mutation and the patient phenotype has been suggested to exist. However, several independent studies have shown that a fraction of NF2 patients with various phenotypes have constitutional deletions that partly or entirely remove one copy of the NF2 gene. The purpose of this study was to examine a 7 Mb interval in the vicinity of the NF2 gene in a large series of NF2 patients in order to determine the frequency and extent of deletions. A total of 116 NF2 patients were analyzed using high-resolution array-comparative genomic hybridization (CGH) on an array covering at least 90% of this region of 22q around the NF2 locus. Deletions, which remove one copy of the entire gene or are predicted to truncate the schwannomin protein, were detected in 8 severe, 10 moderate and 6 mild patients. This result does not support the correlation between the type of mutation affecting the NF2 gene and the disease phenotype. This work also demonstrates the general usefulness of the array-CGH methodology for rapid and comprehensive detection of small (down to 40 kb) heterozygous and/or homozygous deletions occurring in constitutional or tumor-derived DNA.

Adolescent↗
PubMed2001

Adenovirus hexon protein enhances nuclear delivery and increases transgene expression of polyethylenimine/plasmid DNA vectors.

Inefficient nuclear delivery restricts transgene expression using polyelectrolyte DNA vectors. To increase transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexon protein to polyethylenimine (PEI, 800 kDa). Activity of the conjugate was compared with PEI and PEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expression in HepG2 cells than PEI/DNA or complexes containing albumin, without increasing cell uptake. Following cytoplasmic injection into Xenopus laevis oocytes, hexon-containing complexes showed reporter gene expression to be elevated by 10-fold compared with PEI/DNA. The ability of hexon to promote nuclear delivery of PEI/DNA nanoparticles was compared with that of classical nuclear localization sequences (NLS) by measuring transgene expression following intracytoplasmic microinjection of hexon-PEI/DNA complexes and NLS-albumin-PEI/DNA complexes in rat-1 fibroblasts. The resulting nuclear transfer efficiency was in the following order: hexon-PEI/DNA>NLS-albumin-PEI/DNA>PEI/DNA>DNA alone>albumin-PEI/DNA. The activities of both NLS-albumin-PEI and hexon-PEI were abolished by co-injection of wheat germ agglutinin, suggesting that both act by means of the nuclear pore complex (NPC); in contrast, excess free NLS-albumin abolished transgene expression with NLS-albumin-PEI/DNA, but only partially inhibited hexon-PEI/DNA. Nuclear transfer efficiency following cytoplasmic injection was dependent on DNA concentration for all materials, although hexon conjugates showed much better activity than NLS-albumin at low DNA doses (500-1000 plasmids/cell). Our data are consistent with hexon mediating nuclear delivery of plasmid complexes by means of the NPC, using mechanisms that are only partially dependent on the classical NLS import pathway. The hexon-mediated mechanism of nuclear import enables substantially better transgene expression, particularly when DNA concentrations in the cytoplasm are limiting.

Active Transport, Cell Nucleus↗
PubMed2001

Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies.

Adenovirus is a widely used vector for cancer gene therapy because of its high infection efficiency and capacity for transgene expression in both dividing and nondividing cells. However, neutralisation of adenovirus by pre-existing antibodies can lead to inefficient delivery, and the wide tissue distribution of the coxsackie and adenovirus receptor (CAR, the primary receptor for adenovirus type 5) precludes target selectivity. These limitations have largely restricted therapeutic use of adenovirus to local or direct administration. A successful viral gene therapy vector would be protected from neutralising antibodies and exhibit a preferential tropism for target cells. We report here the development of a covalent coating and retargeting strategy using a multivalent hydrophilic polymer based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Incorporation of targeting ligands such as basic fibroblast growth factor and vascular endothelial growth factor on to the polymer-coated virus produces ligand-mediated, CAR-independent binding and uptake into cells bearing appropriate receptors. Retargeted virus is resistant to antibody neutralisation and can infect receptor-positive target cells selectively in mixed culture, and also in xenografts in vivo. Multivalent polymeric modification of adenovirus is an effective way of changing its tropism and interaction with the immune system. As a non-genetic one-step process, the technology is simple, versatile and should yield vectors with an improved safety profile.

Adenoviridae↗
PubMed2001

In vivo gene therapy for colon cancer using adenovirus-mediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase.

Virus-directed enzyme prodrug therapy (VDEPT) utilising cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) into the chemotherapy agent, 5-fluorouracil (5-FU), and has entered into a clinical trial for metastatic colon cancer. To improve this system, a replication-deficient adenovirus, containing a bifunctional fusion gene, CD:uracil phosphoribosyltransferase (UPRT), was constructed (AdCDUPRT). UPRT enhances the conversion of 5-FU into its active metabolites, which inhibit DNA and RNA synthesis. In vitro, AdCDUPRT infection of colon cancer cells resulted in a marked increase in sensitisation to 5-FU, compared with AdCD-infected or uninfected cells. The corollary is a approximately 100-fold and approximately 10 000-fold increase in sensitisation to 5-FC in AdCDUPRT-infected cells, compared to AdCD-infected and uninfected cells, respectively. There was a strong bystander effect in vitro, 70% of tumour cells were killed by 5-FC when only 10% of cells expressed CDUPRT. In vivo, athymic mice with colon cancer xenografts treated with intratumoral AdCDUPRT and intraperitoneal 5-FC, significantly reduced tumour growth rates compared with untreated controls (P = 0.02), whereas AdCD/5-FC treated mice did not. At higher AdCDUPRT virus doses, 5-FC and 5-FU were equally effective at delaying tumour growth compared with controls. In summary, VDEPT for colon cancer utilising AdCDUPRT is more effective than AdCD and the bifunctional CDUPRT gene enables the use of either 5-FC or 5-FU as prodrugs.

Adenoviridae↗
PubMed2000

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase.

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2, 000-fold sensitisation) with 25-50 p.f.u. per cell. Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines. Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001).

Adenoviridae↗
PubMed2000

Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector.

A range of luciferase reporter vectors was constructed, incorporating 5'-flanking sequences from the prostate-specific antigen (PSA), human glandular kallikrein 2 (hKLK2), and cytomegalovirus (CMV) promoters for expression control. Tissue specificity was evaluated in the PSA-positive line LNCaP and PSA-negative cells from different tissues of origin (CoLo320, DG75, EJ, A2780, and Jurkat). The minimal 628-bp PSA and hKLK2 promoters showed only low-level expression in either PSA-positive or PSA-negative cells and showed no increase with the addition of androgen. Tandem duplication of the PSA promoter slightly increased expression in PSA-positive LNCaP cells. The addition of CMV enhancer sequences upstream of a single PSA or hKLK2 promoter substantially but nonspecifically increased luciferase expression in all cell lines tested. However, placing a 1455-bp PSA enhancer sequence upstream of either the PSA or hKLK2 promoters increased expression 20-fold in the PSA-positive cell line LNCaP but not in the PSA-negative lines. Tandem duplication of the PSA enhancer increased expression to approximately 50-fold higher than either promoter alone while retaining tissue-specific control. The level of expression was reduced by the addition of a third copy of the PSA enhancer. Expression from all enhancer constructs was increased 100-fold above basal levels when induced with the androgen dihydrotestosterone, with the PSA-based constructs consistently exhibiting roughly twice the level of expression of the hKLK2-based constructs at all androgen concentrations. Adenovirus vectors were produced in which either enhanced green fluorescent protein or nitroreductase could be expressed from the optimized PSA double enhancer-promoter construct and evaluated in LNCaP cells and the bladder-derived line EJ. Control vectors with the CMV promoter gave good levels of expression in both cell lines, whereas the PSA constructs only produced detectable levels of protein in the LNCaP cells as assessed by fluorescence of enhanced green fluorescent protein or by Western blotting of nitroreductase. LNCaP but not EJ cells were selectively sensitized to the prodrug CB1954 following infection with Ad-PSA(EEP)-NR. The PSA-based nitroreductase virus produced comparable amounts of nitroreductase and sensitization to CB1954 approaching that of the CMV-driven virus. Plasmid and adenovirus constructs combining PSA enhancer and promoter sequences demonstrate selective expression of linked genes in PSA-positive cells. The expression is induced by androgen and gives therapeutically relevant levels of effector proteins.

Adenoviridae↗
PubMed2000

Enhanced proliferation and potassium conductance of Schwann cells isolated from NF2 schwannomas can be reduced by quinidine.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease that is characterized mainly by schwannomas, as well as menigiomas and gliomas. The NF2 gene product merlin/schwannomin acts as a tumor suppressor. Schwann cells derived from NF2 schwannomas showed an enhanced proliferation rate, and electrophysological studies revealed larger K(+) outward currents as compared with controls. Schwann cells isolated from schwannomas of NF2 patients or multiorgan donors were treated with different concentrations of the K(+) current blockers quinidine, tetraethylammonium chloride, and 4-aminopyridine and K(+) outward currents and proliferation rates of these cells were compared. K(+) outward currents of both cell types can be blocked by quinidine. Importantly, treatment with quinidine reduces proliferation of NF2 Schwann cells in a concentration dependent manner but did not reduce proliferation of normal Schwann cells. Therefore, the use of quinidine or quinidine-like components would possibly provide a novel adjuvant therapeutic option for NF2 patients to slow down or freeze growth of schwannomas.

Antineoplastic Agents↗
PubMed2000

The parental origin of new mutations in neurofibromatosis 2.

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder characterized by schwannomas and meningiomas that develop after inactivation of both copies of the NF2 gene. Approximately half of all patients with NF2 have unaffected parents and the disease results from new mutations at the NF2 locus. Loss of heterozygosity (LOH) in tumor specimens due to deletions covering the normal NF2 allele can be used to infer the haplotypes surrounding underlying mutations and determine the allelic origin of new mutations. We studied 71 sporadic NF2 patients using both LOH and pedigree analysis and compared the parental origin of the new mutation with the underlying molecular change. In the 45 informative individuals, 31 mutations (69%) were of paternal and 14 (31%) were of maternal origin (P=0.016). Comparison with corresponding constitutional mutations revealed no correlation between parental origin and the type or location of the mutations. However, in 4 of 6 patients with somatic mosaicism the NF2 mutation was of maternal origin. A slight parent of origin effect on severity of disease was found. Further clinical and molecular studies are needed to determine the basis of these unexpected observations.

Adolescent↗
PubMed2000

Neutralisation of adenovirus infectivity by ascitic fluid from ovarian cancer patients.

Animal models and phase I clinical trials have shown that repeat virus delivery and subsequent transgene expression is limited by the generation of humoral and cellular immune responses directed towards the therapeutic vector. The presence of a pre-existing immune response may even prevent initial delivery. In order to determine the presence of pre-existing anti-adenovirus humoral immunity we analysed ascitic fluid, collected from the peritoneal cavity of patients with advanced ovarian cancer. Twelve ascitic fluid and four matched serum samples were examined. The titre and isotype of anti-adenovirus antibodies was determined by ELISA, and Western blotting identified the molecular basis of the immune response, which was primarily directed towards fibre and penton base. Neutralisation of virus infectivity was assessed in vitro by measurement of green fluorescent protein reporter gene expression. We found that the ascitic fluid samples contain antibodies that recognise both adenovirus types 2 and 5, were predominantly IgG and directed towards the viral antigens responsible for cell adhesion, and had virus neutralising activity.

Adenovirus Infections, Human↗