Biomedical subjects

Hidde J Haisma

Publications and source records attributed to Hidde J Haisma.

At least 19 recordsLinked to original sources

PubMed2007

Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment.

Zinc finger protein transcription factors (ZFP-TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA-binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene-specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA-binding motifs. In this study, we engineered ZFP-TFs to downregulate the activity of the epithelial glycoprotein-2 (EGP-2) promoter. The protein EGP-2 is overexpressed in a wide variety of cancer types and EGP-2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP-2 expression by ZFP-TFs provides a novel anti-cancer therapeutic. Using a straightforward strategy, we engineered a 3-ZFP that could bind a 9 bp sequence within the EGP-2 promoter. After the addition of a repressor domain, this 3-ZFP-TF could efficiently downregulate EGP-2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP-2 promoter activity. To inhibit the endogenous EGP-2 promoter, we engineered 6-ZFP-TFs. Although none of the constructed ZFP-TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP-TFs are versatile bi-directional modulators of gene expression and downregulation of EGP-2 promoter activity using ZFP-TFs can ultimately result in a novel anti-cancer treatment.

Antigens, Neoplasm↗

PubMed2006

A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor.

Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer.

Adenoviridae↗

PubMed2006

Functional inhibition of NF-kappaB signal transduction in alphavbeta3 integrin expressing endothelial cells by using RGD-PEG-modified adenovirus with a mutant IkappaB gene.

In order to selectively block nuclear factor kappaB (NF-kappaB)-dependent signal transduction in angiogenic endothelial cells, we constructed an alphavbeta3 integrin specific adenovirus encoding dominant negative IkappaB (dnIkappaB) as a therapeutic gene. By virtue of RGD modification of the PEGylated virus, the specificity of the cell entry pathway of adenovirus shifted from coxsacki-adenovirus receptor dependent to alphavbeta3 integrin dependent entry. The therapeutic outcome of delivery of the transgene into endothelial cells was determined by analysis of cellular responsiveness to tumor necrosis factor (TNF)-alpha. Using real time reverse transcription PCR, mRNA levels of the cell adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, the cytokines/growth factors IL-6, IL-8 and vascular endothelial growth factor (VEGF)-A, and the receptor tyrosine kinase Tie-2 were assessed. Furthermore, levels of ICAM-1 protein were determined by flow cytometric analysis. RGD-targeted adenovirus delivered the dnIkappaB via alphavbeta3 to become functionally expressed, leading to complete abolishment of TNF-alpha-induced up-regulation of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, VEGF-A and Tie-2. The approach of targeted delivery of dnIkappaB into endothelial cells presented here can be employed for diseases such as rheumatoid arthritis and inflammatory bowel disease where activation of NF-kappaB activity should be locally restored to basal levels in the endothelium.

Adenoviridae↗

PubMed2006

Step into the groove: engineered transcription factors as modulators of gene expression.

Increasing knowledge about the influence of dysregulated gene expression in causing numerous diseases opens up new possibilities for the development of innovative therapeutics. In this chapter, we first describe different mechanisms of misregulated gene expression resulting in various pathophysiological conditions. Then, an overview is given of different technologies developed to readjust expression levels of genes. One of the most promising upcoming approaches in this respect is the development of engineered zinc-finger transcription factors. Results obtained from modulating endogenous gene expression using such engineered transcription factors are reviewed in depth. Finally, we address possible pitfalls of using such transcriptional targeting approaches at the "chromatin level." We describe aspects of studies at this level that influence successful DNA binding of engineered transcription factors, thereby affecting gene activity. Engineered transcription factors have great promise as potent therapeutics. Moreover, this technology is expected to yield fundamental knowledge about the organization and function of the genome.

Animals↗

PubMed2006

Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation.

Lymphatic vessel plasticity and stability are of considerable importance when attempting to treat diseases associated with the lymphatic vasculature. Development of lymphatic vessels during embryogenesis is dependent on vascular endothelial growth factor (VEGF)-C but not VEGF-D. Using a recombinant adenovirus encoding a soluble form of their receptor VEGFR-3 (AdVEGFR-3-Ig), we studied lymphatic vessel dependency on VEGF-C and VEGF-D induced VEGFR-3 signaling in postnatal and adult mice. Transduction with AdVEGFR-3-Ig led to regression of lymphatic capillaries and medium-sized lymphatic vessels in mice under 2 weeks of age without affecting collecting lymphatic vessels or the blood vasculature. No effect was observed after this period. The lymphatic capillaries of neonatal mice also regressed partially in response to recombinant VEGFR-3-Ig or blocking antibodies against VEGFR-3, but not to adenovirus-encoded VEGFR-2-Ig. Despite sustained inhibitory VEGFR-3-Ig levels, lymphatic vessel regrowth was observed at 4 weeks of age. Interestingly, whereas transgenic expression of VEGF-C in the skin induced lymphatic hyperplasia even during embryogenesis, similar expression of VEGF-D resulted in lymphangiogenesis predominantly after birth. These results indicate considerable plasticity of lymphatic vessels during the early postnatal period but not thereafter, suggesting that anti-lymphangiogenic therapy can be safely applied in adults.

Adenoviridae↗

PubMed2005

Ovarian cancer targeted adenoviral-mediated mda-7/IL-24 gene therapy.

OBJECTIVE: We have previously shown that adenoviral-mediated melanoma differentiation-associated gene-7 (Ad.mda-7) therapy induces apoptosis in ovarian cancer cells. However, the apoptosis induction was low and directly correlated with infectivity of Ad.mda-7. The objective of this study was to derive ovarian cancer targeted infectivity-enhanced adenoviral vectors encoding mda-7 and evaluate their enhancement in therapeutic efficacy for ovarian carcinoma. METHODS: Infectivity-enhanced adenoviral vectors encoding mda-7 Ad.RGD.mda-7 and Ad.RGD.pK7.mda-7 were derived by incorporation of RGD and or RGD and Pk7 motifs in the fiber knobs by genetic modification. Viruses were validated by PCR for presence of mda-7 and by Western blot for expression of MDA-7 protein. To test the enhancement of therapeutic efficacy of these viruses, a panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1, were infected by either Ad.mda-7 or Ad.RGD.mda-7 and Ad.RGD.pK7.mda-7 or their respective control viruses and the cell killing was evaluated by crystal violet staining in vitro. Further, therapeutic efficacy was evaluated in vivo using human ovarian cancer xenograft mouse models. RESULTS: Both Ad.RGD.pK7.mda-7 and Ad.RGD.mda-7 showed significant increase in cell killing in vitro compared to unmodified Ad.mda-7 with Ad.RGD.pK7.mda-7 showing highest cell killing. Further, Ad.RGD.pK7.mda-7 showed a significant increase in survival of mice bearing human ovarian cancer xenografts compared to Ad.mda-7 and other control groups. CONCLUSION: Infectivity-enhanced Ad.RGD.mda-7 and Ad.RGD.pK7.mda-7 viruses significantly enhanced ovarian cancer tumor cell killing in vitro. Significant prolongation of survival by Ad.RGD.pK7.mda-7 in murine ovarian cancer models demonstrates the high clinical translational potential of these viruses for ovarian cancer therapy.

Adenocarcinoma↗

PubMed2005

Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command.

Modulating gene expression directly at the DNA level represents a novel approach to control cellular processes. In this respect, zinc finger protein DNA-binding domains can be engineered to target virtually any gene. Coupling of a transcription activation or repression domain to these zinc fingers permits regulating gene expression at will, providing a platform of unlimited therapeutic applications. In this review, steps involved in the engineering of zinc finger protein transcription factors are described. In addition, an overview of endogenous genes successfully targeted for modulating expression by engineered zinc finger protein transcription factors is given. So far, research has mainly focused on targeting genes involved in cancer and angiogenesis, with encouraging evaluation in vivo and progression into a clinical trial. Altogether, engineered zinc finger proteins offer a new and exciting direction in the field of medical research with promising prospects.

Animals↗

PubMed2005

Adenovirus-mediated gene transfer of placental growth factor to perivascular tissue induces angiogenesis via upregulation of the expression of endogenous vascular endothelial growth factor-A.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family that binds specifically to VEGF receptor (VEGFR)-1. However, the mechanism of PlGF- and VEGFR-1-mediated angiogenesis has remained unclear and some in vitro studies suggest that VEGF-A/VEGFR-2 signaling may also play a role in PlGF-mediated angiogenesis. To clarify these issues we evaluated angiogenic responses in a well-characterized periadventitial angiogenesis model using adenovirus-mediated PlGF-2 (AdvPlGF-2) gene transfer. We also investigated the roles of VEGFR-1 and VEGFR-2 in PlGF-2-mediated angiogenesis. Using a periadventitial collar technique, AdvPlGF-2 (1 x 10(9) plaque-forming units/ml) was transferred to the adventitia of New Zealand White rabbits alone or together with adenoviruses encoding soluble VEGFR-1 (sVEGFR-1) or soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were killed 7 days after gene transfer. Increased neo-vessel formation, upregulation of endogenous VEGF-A expression, and a significant inflammatory response were seen in AdvPlGF-2-transduced arteries. The neo-vessels were large and well perfused. sVEGFR-1 and sVEGFR-2 suppressed the angiogenic response of PlGF-2 by 80 and 71.7%, respectively. We conclude that adenovirus-mediated PlGF-2 gene transfer to vascular tissue increases endogenous VEGF-A expression and produces significant angiogenesis. Both sVEGFR-1 and sVEGFR-2 can inhibit PlGF-2-mediated angiogenesis. PlGF-2 is a potentially useful candidate for the induction of therapeutic angiogenesis in vivo.

Adenoviridae↗

PubMed2005

The human norepinephrine transporter in combination with 11C-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy.

UNLABELLED: Although the herpes simplex virus thymidine kinase gene has been frequently applied as a reporter gene for monitoring gene transfection in animals, it has some intrinsic limitations for use in humans. In our search for a reporter gene that lacks these limitations, we have evaluated the feasibility of the human norepinephrine transporter (hNET) as a reporter gene in combination with the reporter probe 11C-m-hydroxyephedrine (mHED) for PET. METHODS: An adenoviral vector (AdTrack-hNET) containing the hNET gene as reporter gene and the enhanced green fluorescent protein (EGFP) as a substitute for a therapeutic gene was constructed. After COS-7, A2780, and U373 cells were transiently transduced with AdTrack-hNET, hNET protein expression, EGFP fluorescence, and cellular uptake of 11C-mHED were determined. In rats, U373 tumor xenografts were grown and transiently transduced with either AdTrack-hNET or an AdTrack-Luc control adenovirus. Intratumoral accumulation of 11C-mHED was determined by PET and ex vivo biodistribution. The tumors were subsequently examined for EGFP fluorescence. RESULTS: 11C-mHED uptake was positively correlated with AdTrack-hNET viral titer and hNET protein expression. However, large differences in transfection efficiency between cell lines were observed. The highest 11C-mHED uptake was found in hNET transfected U373 cells, in which tracer uptake was >70-fold higher than that in control cells. 11C-mHED accumulation could be inhibited by desipramine, a potent inhibitor of hNET. In all cell lines, 11C-mHED uptake was positively correlated with EGFP fluorescence, implying that imaging of hNET with 11C-mHED would enable monitoring of a coexpressed therapeutic gene. In the animal model, gene transfection efficiencies were very low, as determined by EGFP fluorescence. Still, a significantly higher 11C-mHED uptake in hNET transduced tumors than that in control tumors was demonstrated by ex vivo biodistribution studies. PET with a clinical camera could visualize 1 of 3 hNET transduced tumors, indicating that the transfection efficiency was near the detection limit. CONCLUSION: These results indicate that monitoring of gene therapy using the hNET/11C-mHED reporter gene/probe is feasible, but further investigation with regard to the sensitivity of the technique is required.

Adenoviridae↗

PubMed2004

A novel ex vivo model system for evaluation of conditionally replicative adenoviruses therapeutic efficacy and toxicity.

PURPOSE: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver. EXPERIMENTAL DESIGN: The Krumdieck thin-slice tissue culture system was used to obtain and culture slices of tumor xenografts of ovarian cancer cell lines, human primary ovarian tumors, and human liver. We determined the viability of slices in culture over a period of 36 to 48 hours by ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt)]) (MTS) assay. In vitro Hey cells, slices of Hey xenografts, and human ovarian tumor or human liver slices were infected with 500vp/cell of either replication competent wild-type adenovirus (Ad5/3wt), conditionally replicative adenovirus (Ad5/3cox-2), or the replication deficient adenovirus (Ad5/3luc1). At 12-, 24-, and 36-hour intervals, the replication of adenoviruses in these slices was determined by quantitative reverse transcription-PCR of adenoviral E4 copy number. RESULTS: Primary tumor slices were able to maintain viability for up to 48 hours after infection with nonreplicative virus (Ad5luc1). Infection of Hey xenografts with Ad5/3cox-2 showed replication consistent with that seen in Hey cells infected in an in vitro setting. Primary tumor slices showed replication of both Ad5/3wt and Ad5/3cox over a 36-hour time period. Human liver slices showed replication of Ad5/3wt but a relative reduction in replication of Ad5/3cox-2 indicative of conditional replication "liver off" phenotype, thus predicting lower toxicity. CONCLUSIONS: The thin-slice model system represents a stringent method of ex vivo evaluation of novel replicative adenoviral vectors and allows assessment of human liver replication relative to human tumor replication. This is the first study to incorporate this system for evaluation of therapeutic efficacy and replicative specificity of conditionally replicative adenoviruses. Also, the study is the first to provide a valid means for preclinical assay of potential conditionally replicative adenovirus-based hepatotoxicities, thus providing a powerful tool to determine therapeutic index for clinical translation of conditionally replicative adenoviruses.

Adenoviridae↗

PubMed2004

A methylester of the glucuronide prodrug DOX-GA3 for improvement of tumor-selective chemotherapy.

The glucuronide prodrug of doxorubicin, DOX-GA3, can be selectively activated in tumors by extracellular human beta-glucuronidase, resulting in a better therapeutic index than doxorubicin. DOX-GA3, however, is rapidly excreted by the kidney. We hypothesized that slow release of DOX-GA3 from its methylester, DOX-mGA3, by esterase activity in blood would result in improved circulation half-life (t(1/2)) of DOX-GA3. DOX-mGA3 was synthesized more efficiently with an overall yield of 60% as compared to 37% in the case of DOX-GA3. We showed that DOX-mGA3 was enzymatically converted to DOX-GA3 with a t(1/2) of approximately 0.5 min in mouse plasma to 2.5 h in human plasma, which was in agreement with differences in esterase activity between species. DOX-mGA3, similar to DOX-GA3, was at least 37-fold less potent than the parent drug doxorubicin in growth inhibition of four different human malignant cell lines in vitro. Incubation of OVCAR-3 cells with DOX-mGA3 in combination with an excess of human beta-glucuronidase (0.05 U mL(-1)) resulted in a similar growth inhibition to that of doxorubicin. Intravenous administration of DOX-mGA3 in FMa-bearing mice resulted in an area under the concentration versus time curve (AUC) of DOX-GA3 in tumor and most normal tissues that was 2.5- to 3-fold higher than after the same dose of DOX-GA3 itself. In tumor tissue, this was accompanied by a 2.7-fold increase in the AUC of doxorubicin from DOX-mGA3 than from DOX-GA3. In conclusion, an advantage of DOX-mGA3 over DOX-GA3 is that this prodrug can be produced with a higher yield. Another important advantage is the improved pharmacokinetics of the lipophilic DOX-mGA3 as compared to that of the hydrophilic DOX-GA3. This effect may even be more pronounced in man, because of the lower plasma esterase activity than measured in mice.

Animals↗

PubMed2004

Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine-conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco-conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol-3 kinase pathway (PI3K) and nuclear factor-kappaB were inhibited using LY 294002 and adenoviral overexpression of dominant-negative IkappaB, respectively. The role of p38 and extracellular signal-regulated protein kinase mitogen-activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin-D. Apoptosis was determined by measuring caspase-3, -9, and -8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase-8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA-induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF-kappaB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA-induced mitochondria-controlled apoptosis by activating survival pathways.

Animals↗

PubMed2004

Infectivity enhanced adenoviral-mediated mda-7/IL-24 gene therapy for ovarian carcinoma.

OBJECTIVE: Melanoma differentiation associated gene-7 [mda-7/Interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the anti-tumor effect of adenovirus-mediated mda-7/IL-24 (Ad.mda-7) gene therapy in ovarian carcinoma and further improve anti-tumor effect by enhancing infectivity of Ad.mda-7. METHODS: A panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1 and control normal human mesothelial cells, were infected by a replication deficient recombinant adenovirus encoding mda-7/IL-24 and control virus Ad.CMV.Luc. After 72 h, apoptosis was evaluated by TUNEL and Hoechst staining and further quantified by fluorescent activated cell sorter (FACS) analysis. Infectivity of Ad.mda-7 was enhanced by retargeting it to CD40 or EGF receptors overexpressed on ovarian cancer cells. Subsequently, enhancement in apoptosis of CD40- or epidermal growth factor receptor (EGFR)-retargeted Ad.mda-7 was evaluated. RESULTS: Adenoviral-mediated delivery of mda-7 induces apoptosis ranging from 10-23% in human ovarian cancer cells tested with the highest percentage of apoptosis noted in SKOV3 cells. Minimal apoptosis was noted in normal mesothelial cells. CD40- or EGFR-retargeted Ad.mda-7 increased apoptosis by 10-32% when compared to that achieved with untargeted Ad.mda-7. CONCLUSION: Ad.mda-7 exhibits ovarian cancer-specific apoptosis, but does not affect normal human mesothelial cells. Infectivity enhanced CD40- and EGFR-retargeted Ad.mda-7 augments apoptosis induction, thus increasing the therapeutic index and translational potential of Ad.mda-7 gene therapy.

Adenoviruses, Human↗

PubMed2004

Pronounced antitumor efficacy by extracellular activation of a doxorubicin-glucuronide prodrug after adenoviral vector-mediated expression of a human antibody-enzyme fusion protein.

Tumor-specific activation of the glucuronide prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate (DOX-GA3), by beta-glucuronidase present in necrotic tumor areas might be improved after transduction of tumor cells to secrete a targeted form of beta-glucuronidase. To that end, we constructed an adenovirus vector, designated Ad/C28-GUSh, encoding human beta-glucuronidase fused to a human single-chain Fv (scFv) against the epithelial cell adhesion molecule (EpCAM), C28, and preceded by a signal sequence for secretion. Antibody specificity and enzyme activity were retained in the fusion protein secreted by tumor cells infected with Ad/C28-GUSh. Diffusion of fusion protein from transduced tumor cells within MCF-7 multicellular spheroids was visualized by immunohistochemistry. Treatment of spheroids with Ad/C28-GUSh and DOX-GA3 resulted in growth inhibition comparable to treatment with doxorubicin alone. Treatment of well-established FMa human ovarian cancer xenografts with intravenous injection of DOX-GA3 (500 mg/kg) resulted in a tumor volume-doubling time of 23.8 days compared to 8.0 days for phosphate-buffered saline (PBS)-treated mice. Intratumoral administration of Ad/C28-GUSh before DOX-GA3 enhanced the growth inhibition and increased the tumor volume-doubling time to 43.1 days (p < 0.01), while virus alone had no effect. Thus, we have successfully shown that an adenovirus vector encoding a secreted, targeted form of human beta-glucuronidase can further improve DOX-GA3 monotherapy.

Adenoviridae↗

PubMed2004

A novel strategy to modify adenovirus tropism and enhance transgene delivery to activated vascular endothelial cells in vitro and in vivo.

To assess the possibilities of retargeting adenovirus to activated endothelial cells, we conjugated bifunctional polyethylene glycol (PEG) onto the adenoviral capsid to inhibit the interaction between viral knob and coxsackie-adenovirus receptor (CAR). Subsequently, we introduced an alphav integrin-specific RGD peptide or E-selectin-specific antibody to the other functional group of the PEG molecule for the retargeting of the adenovirus to activated endothelial cells. In vitro studies showed that this approach resulted in the elimination of transgene transfer into CAR-positive cells, while at the same time specific transgene transfer to activated endothelial cells was achieved. PEGylated, retargeted adenovirus showed longer persistence in the blood circulation with area under plasma concentration-time curve (AUC) values increasing 12-fold compared to unmodified virus. Anti-E-selectin antibody-PEG-adenovirus selectively homed to inflamed skin in mice with a delayed-type hypersensitivity (DTH) inflammation, resulting in local expression of the reporter transgene luciferase. This is the first study showing the benefits of PEGylation on adenovirus behavior upon systemic administration. The approach described here can form the basis for further development of adenoviral gene therapy vectors with improved pharmacokinetics and increased efficiency and specificity of therapeutic gene transfer into endothelial cells in disease.

Adenoviridae↗

PubMed2003

Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities.

Designer molecules that can specifically target pre-determined DNA sequences provide a means to modulate endogenous gene function. Different classes of sequence-specific DNA-binding agents have been developed, including triplex-forming molecules, synthetic polyamides and designer zinc finger proteins. These different types of designer molecules with their different principles of engineered sequence specificity are reviewed in this paper. Furthermore, we explore and discuss the potential of these molecules as therapeutic modulators of endogenous gene function, focusing on modulation by stable gene modification and by regulation of gene transcription.

Animals↗

PubMed2003

CD40-targeted adenoviral gene transfer to dendritic cells through the use of a novel bispecific single-chain Fv antibody enhances cytotoxic T cell activation.

Adenoviral (Ad) transduction of dendritic cells (DC) is a promising vaccination strategy. However, clinical applicability of Ad vectors is hampered by the necessity to use high titers of infectious Ad particles for efficient DC transduction. Here, we report on the production of a bacterially expressed bispecific conjugate, consisting of a fusion of recombinant single-chain (sc) mAb Fv fragments, which bind and neutralize the Ad fiber knob (through the S11 mAb scFv) and retarget Ad to CD40 on the DC surface (through the G28-5 mAb scFv). We show that this bispecific scFv fusion protein significantly enhances transduction efficiency of monocyte-derived DC (MoDC), reduces the amount of virus needed for a given level of transduction, and increases the ability of MoDC to activate CTL in an antigen specific manner. This single-component conjugate may prove to be a valuable immunotherapeutic tool for the targeting of Ad to DC in vivo.

Adenoviridae↗

PubMed2003

Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin.

Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA3) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present study, we established that cellular uptake of DNR-GlA3 and DNR-GsA3 was low in contrast to that of daunorubicin. Recombinant human beta-glycosidase converted both prodrugs to daunorubicin as shown by liquid chromatography. The kinetics of the conversion of DNR-GlA3 and DNR-GsA3 by human beta-glycosidase, however, was unfavorable as the K(m) values were, respectively, 3- and 6-fold higher than those of another mammalian beta-glycosidase of bovine origin. The V(max) values were, respectively, 3.3 and 8.5nmol/hr/mg as compared to 158.3 and 147.8nmol/hr/mg of the bovine enzyme. Treatment of OVCAR-3 cells with human beta-glycosidase (0.5U/mL) and 0.5 microM DNR-GlA3 or DNR-GsA3 resulted in, respectively, 86 and 81% cell growth inhibition, while the prodrugs alone inhibited growth to only 19 and 1%. Treatment of cells with the bovine enzyme and the prodrugs inhibited cell growth more efficiently. We conclude that the endogenous intracellular beta-glycosidase is not available for extracellular prodrug activation. Thus, the incorporation of the enzyme in enzyme-prodrug therapy might be an elegant approach to achieve tumor-specific prodrug conversion. The efficiency of glycoside prodrug conversion might be improved by design of a prodrug that is more readily activated by human beta-glycosidase or by evolution of the enzyme into a mutant form that displays high activity towards these prodrugs.

Animals↗