Michael Maris

FK506 reduces neuroinflammation and dopaminergic neurodegeneration in an α-synuclein-based rat model for Parkinson's disease.

Alpha-synuclein (α-synuclein) is considered a key player in Parkinsons disease (PD), but the exact relationship between α-synuclein aggregation and dopaminergic neurodegeneration remains unresolved. There is increasing evidence that neuroinflammatory processes are closely linked to dopaminergic cell death, but whether the inflammatory process is causally involved in PD or rather reflects secondary consequences of nigrostriatal pathway injury is still under debate. We evaluated the therapeutic effect of the immunophilin ligand FK506 in a rAAV2/7 α-synuclein overexpression rat model. Treatment with FK506 significantly increased the survival of dopaminergic neurons in a dose-dependent manner. No reduction in α-synuclein aggregation was apparent in this time window, but FK506 significantly lowered the infiltration of both T helper and cytotoxic T cells and the number and subtype of microglia and macrophages. These data suggest that the anti-inflammatory properties of FK506 decrease neurodegeneration in this α-synuclein-based PD model, pointing to a causal role of neuroinflammation in the pathogenesis of PD.

Optimization of Multimodal Imaging of Mesenchymal Stem Cells Using the Human Sodium Iodide Symporter for PET and Cerenkov Luminescence Imaging

Purpose The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters. Methods First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, 124I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using 99mTcO4 − radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cells differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI. Results The expression of both imaging reporter genes was functional and specific. An elution of 99mTcO4 − from the cells was observed, with 31% retention after 3 h. After labeling cells with 124I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using 124I small-animal PET, CLI and BLI. Conclusions This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.

Immunological Ignorance Allows Long-Term Gene Expression After Perinatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer to Murine Airways

Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and α(1)-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months.

Cereulide food toxin, beta cell function and diabetes: Facts and hypotheses

The incidence of both type 1 and type 2 diabetes is increasing and although environmental pollutants are believed to be potential culprits, the extent to which they can be held responsible remains uncertain. Some bacterial strains of the Bacillus cereus produce a toxin, cereulide, which is frequently found in starchy meals and which is difficult to eradicate from the food chain as it is highly resistant to heat, acidity and proteolysis. While cereulide is well known to cause acute emetic toxicity when ingested at high doses, several in vitro studies have shown that also extremely low doses of cereulide can be toxic, with beta cells being particularly sensitive. Mechanistically, such low doses impair the mitochondrial activity of the beta cells thereby leading to hampered insulin secretion and cell death, both key traits in the pathophysiology of diabetes. In vivo studies of chronic or repeated low dose exposure to cereulide are currently lacking, but should be performed to further clarify the true relevance of cereulide as a potential environmental contributor to the ongoing diabetes epidemic.

413. Development and Characterization of Lentiviral Vectors for Hemophilia Gene Therapy

Monogenic inherited diseases can potentially be cured by gene therapy, which has already proven to be successful in a number of preclinical and clinical studies. A major target for gene therapy is the liver, since many proteins are synthesized in hepatocytes and secreted proteins can readily be delivered into the bloodstream. Lentiviral vectors are attractive vectors for hepatic gene delivery since they stably integrate into the target cell genome and thus can potentially give rise to life-long expression of the therapeutic protein (VandenDriessche et al., Blood 100:813–822, 2002). Lentiviral vectors do not express any potentially immunogenic viral genes and induce only minimal and transient liver toxicity and no pro-inflammatory cytokines (IL-6). However, intravenous delivery of a lentiviral vector pseudotyped with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G), resulted in efficient transduction of antigen presenting cells (APCs), primarily in liver and spleen, and transgene expression in APCs when a ubiquitously expressed cytomegalovirus (CMV) immediate early promoter was used. This increased the risk of developing a humoral immune response directed against the transgene product. Obviously, to obtain long-term expression, such an immune reaction has to be avoided. Different strategies are being pursued to direct expression of the transgene exclusively in hepatocytes and to prevent gene expression in APCs. We have therefore generated lentiviral vectors containing different hepatocyte-specific expression cassettes, which were titered by an optimized Q-PCR approach. Expression of coagulation factor IX using a human alpha1-antitrypsin promoter in conjunction with an ApoE enhancer repeat and a matrix attachment region (MAR) resulted in therapeutic FIX expression levels in vivo that were as high as when a CMV promoter were used. An alternative strategy to redirect expression to hepatocytes is to increase hepatocyte-specific transduction, while reducing vector uptake by APCs. This could potentially be achieved by pseudotyping the lentiviral vectors with alternative envelope proteins. Another potential advantage of reducing vector uptake by APCs, is that it may reduce the total vector dose necessary to reach a certain expression level. In a separate study with high-capacity adenoviral (HC-Ad) vectors, 10-fold higher expression levels were obtained after temporary depletion of macrophages (Chuah et al., Blood 101:1734–1743, 2003). However, the effect of transient APC depletion on transgene expression levels was not as dramatic following lentiviral gene transfer, suggesting that lentiviral vectors result in reduced APC transduction compared to when HC-Ad vectors are used. To further improve the safety profile of lentiviral vectors, purification of vector preparations is being optimized by ion-exchange chromatography. In summary, lentiviral vectors for hepatic gene delivery are being improved further at multiple levels by (i) designing more potent and hepatocyte-specific expression cassettes; (ii) developing alternative pseudotypes and (iii) implementing improved vector purification methods.