Expression Of Beta-galactosidase Transgene Research Articles

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in beta-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The 'cassette' design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

Fibrin scaffold promotes adenoviral gene transfer and controlled vector delivery

Gene therapy using adenoviral vectors in tissue regeneration is hindered by a short duration of transgene expression. It is hypothesized that a fibrin scaffold will enhance delivery of the adenovirus to a wound site, precluding the need for high repeated doses. It was aimed to analyze whether fibrin could deliver a low single dose of viral vector to a wound site, without compromising transfection efficiency. Fibrin scaffold containing adenovirus encoding beta-galactosidase, fibrin alone, adenovirus alone, and no treatment groups were applied to a rabbit ear ulcer model. beta-Galactosidase transgene expression was measured at 7 and 14 days. Transgene expression was enhanced in the fibrin containing adenovirus group at 7 days. By 14 days, there was low expression and no difference between groups. Stereological methods assessing wound healing aimed to determine whether the adenovirus capsid elicited an unfavorable inflammatory response and whether fibrin's beneficial properties were altered by addition of adenovirus. The fibrin adenovirus group showed a wound-healing response similar to fibrin alone, showing maximum cellularity and angiogenesis at 7 days. By 14 days, cellularity and angiogenesis subsided, and this effect was not inhibited by the presence of adenovirus. Adenovirus alone did not cause an unfavorable inflammatory response. It is concluded the fibrin aids in the delivery of a low-dose viral vector, thereby avoiding a chronic inflammatory response, and allowing superior transfection than viral vector alone. This has wide-ranging implications on the use of viral vectors in tissue engineering.

Bone marrow‐derived myofibroblasts contribute functionally to scar formation after myocardial infarction

Myofibroblasts play a major role in scar formation during wound healing after myocardial infarction (MI). Their origin has been thought to be interstitial cardiac fibroblasts. However, the bone marrow (BM) can be a source of myofibroblasts in a number of organs after injury. We have studied the temporal, quantitative and functional role of BM-derived (BMD) myofibroblasts in myocardial scar formation. MI was induced by permanent coronary artery ligation in mice reconstituted with EGFP or pro-Col1A2 transgenic BM. In the latter, luciferase and beta-galactosidase transgene expression mirrors that of the endogenous pro-collagen 1A2 gene, which allows for functional assessment of the recruited cells. After MI, alpha-SMA-positive myofibroblasts and collagen I gradually increased in the infarct area until day 14 and remained constant afterwards. Numerous EGFP-positive BMD cells were present during the first week post-MI, and gradually decreased afterwards until day 28. Peak numbers of BMD myofibroblasts, co-expressing EGFP and alpha-SMA, were found on day 7 post-MI. An average of 21% of the BMD cells in the infarct area were myofibroblasts. These cells constituted up to 24% of all myofibroblasts present. By in vivo IVIS imaging, BMD myofibroblasts were found to be active for collagen I production and their presence was confined to the infarct area. These results show that BMD myofibroblasts participate actively in scar formation after MI.

Novel dextran–spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

Recently, a novel cationic polymer, dextran-spermine (D-SPM) was developed for gene delivery. An efficient transfection was obtained using this polycation for a variety of genes and cell lines in serum-free or serum-poor medium. However, transfection using the water-soluble D-SPM-based polyplexes decreased with increasing serum concentration in cell culture in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium. In order to overcome this obstacle, oleyl derivatives of D-SPM (which form micelles in aqueous phase) were synthesized at 1, 10, and 20 mol% of oleyl moiety to polymer epsilon-NH2 to form N-oleyl-D-SPM (ODS). Polyplexes based on ODS transfected well in medium containing 50% serum. Comparison with polyplexes based on well-established polymers (branched and linear polyethyleneimine) and with DOTAP/Cholesterol lipoplexes showed that regarding beta-galactosidase transgene expression level and cytotoxicity in tissue culture, the D-SPM and ODS compare well with the above polyplexes and lipoplexes. Intracellular trafficking using FITC-labeled ODS and Rhodamine-labeled pGeneGrip plasmid cloned with hBMP2 monitored by confocal microscopy revealed that during the transfection process the fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus, while the cell cytoplasm was free of fluorescent particles, suggesting that the polyplexes move in the cell toward the nucleus by vesicular transport through the cytoplasm and not by a random diffusion. We found that the plasmids penetrate the cell nucleus without the polymer. Preliminary results in zebra fish and mice demonstrate the potential of ODS to serve as an efficient nonviral vector for in vivo transfection.

Adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective IL-4 to murine synovium.

Treatments for rheumatoid arthritis and other inflammatory arthropathies are often ineffective at preventing joint destruction. Long-term genetic modification of the cells lining the joint space (synoviocytes) in vivo represents a potential method for the treatment of these chronic conditions. However, a vector capable of efficiently transducing synoviocytes in vivo for a persistent period has not been available. The present report describes the genetic modification of synoviocytes in vivo using recombinant adeno-associated virus. High-titer adeno-associated virus encoding the gene for Escherichia coli beta-galactosidase was injected into the knee joints of mice. Synovial tissues were then examined for beta-galactosidase transgene expression by in situ staining and by fluorometry. High-efficiency, persistent transgene expression was observed in the synovium with no evidence of vector-induced inflammation. Expression was observed for at least 7 months and was higher in arthritic than nonarthritic mice. Gene transfer of murine IL-4 to the joints of mice with collagen-induced arthritis led to detectable levels of IL-4 in the joint and protection from articular cartilage destruction. These data suggest that adeno-associated virus may be a useful vector for gene delivery to the synovium for the treatment of inflammatory arthropathies.

Foetal gene delivery in mice by intra-amniotic administration of retroviral producer cells and adenovirus.

With the aim of developing foetal gene therapy for cystic fibrosis, we have investigated the possibility of gene targeting to the mouse foetus with two different viral vector systems and at different times of gestation. We report here that recombinant retrovirus producing cells administered into the intra-amniotic cavity of mid- to late-gestation mouse MF1 foetuses survive in the amniotic fluid and are able to engraft to a certain extent in foetal tissues. By production of infectious virus they mediate transduction and beta-galactosidase transgene expression in neighbouring foetal tissues 24 to 72 h following injection. Retrovirus producer cells could, therefore, become a means to overcome the limitations of low retroviral titre, for in vivo foetal gene transfer. To investigate the developmental stage at which transduction of the airways and enteral systems can be obtained we also administered a highly infective first generation adenoviral vector (AdRSV beta gal) into the amniotic cavity of foetal mice between 13 to 16 days post coitus, beta-galactosidase activity was detected between 24 to 120 h after injection. The highest levels of transgene expression were generally observed between 48 to 72 h following injection of the adenoviral vector. We demonstrate that infection of the pulmonary airways is dependent on the developmental stage of the foetus and can be achieved on the 15th day of gestation.