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Beta-cell neogenesis is an attractive option for restoring beta-cell mass in diabetes. Recent findings indicate that beta-cells can be derived from alpha-cells under conditions of extreme beta-cell loss in mice. However, little is known about the potential of transdifferentiation capacity of human islet cells.

Islets transplantation may restore insulin production and normoglycemia in type 1 diabetic recipients. However, the efficacy of transplantation is seriously compromised by immune memory, autoreactive T-cells, and allograft rejection.

To study and evaluate the potential of gene therapy for the treatment of hemophilia A, we have developed a retroviral vector system for the introduction and expression of factor VIII cDNA in somatic cells in vivo Bone-marrow cells were infected with the recombinant retrovirus. Transplantation of infected bone-marrow cells into lethally irradiated mice resulted in the formation of spleen colonies. Although the presence of the vector provirus could be detected in the genomes of the stem-cell-derived cells, no synthesis of human factor VIII or its RNA could be detected. As an alternative approach, factor-VIII-producing fibroblasts of human and murine origin were implanted into athymic nude mice, embedded in an artificial collagen matrix. In the case of human skin fibroblasts, cells isolated from the grafts 4 weeks after implantation still have the capacity to secrete factor VIII when regrown in culture. However, no human factor VIII could be detected in the plasma of the recipient mice. From these data we conclude that retroviral vectors can be used for gene transfer into somatic cells of laboratory animals in vivo. The cause of the apparent lack of expression will be discussed.

Background: New promoters for high level short-term glia-specific expression have been created. These 'super' promoters are based on the promoter of the gene for glial fibrillary acidic protein (GFAP). Addition of three extra copies of the glia-specific enhancer elements A, B and D to the glia-specific 2.2 kb GFAP-promoter fragment (gfa2) resulted in 2 log higher expression levels after plasmid transfections. We describe the characteristics of these promoters in the context of the Ad5 E1 region in vitro and in vivo.

Specificity of gene delivery is a key prerequisite for gene therapy. We have previously demonstrated that modification of the minor capsid protein IX (pIX) can be used to modify cell type specificity of human adenovirus type-5 (HAdV-5) particles. The 14.3 kDa hexon-associated pIX is strongly conserved between the different members of the mastadenoviruses. Protein IX is believed to function as a capsid cement by linking the hexons in groups-of-nine (GONs). Although viruses lacking pIX do not form GONs, and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine-zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains the putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. Further analysis of these mutants revealed that deletion of the putative leucine-zipper domain does not affect capsid stabilization by pIX, demonstrating that trimerization is not necessary for the heat-stability phenotype1. To facilitate pseudotyping adenoviral vectors with pIX variants we have set up an efficient system for the generation of pIX-expressing helper cell lines. With an optimized pIX-expression cassette, we generated monoclonal and polyclonal helper cell lines that express wt as well as modified pIX genes to levels equivalent as in a wt HAdV-5 infected cells. Immune-affinity electron microscopy on viruses lacking the pIX gene demonstrated that more than 96% of the particles contain pIX protein incorporated in their capsids after propagation on the pIX-expressing helper cell lines. In addition, the pIX amounts in the helper cells are sufficient to generate heat-stable particles. Finally, the ratio between the pIX protein and other capsid proteins is identical to those found in wt particles. The cell lines are very stable: cell lines grown for two months in the absence of selection contain pIX in amounts identical to the parental line. This demonstrates that pIX is not toxic to cells. These data, together with our previous demonstration that alpha-helical spacers can be fused to pIX to expose new ligands on the outer surface of particles2, establishes that modification of pIX is feasible for pseudotyping adenovirus vectors.

In mastadenoviruses, the hexon-associated protein IX is a 14.3kDa protein that is strongly conserved between the different members of the genus. It associates with nine hexon capsomers to form group-of-nine hexons (GON). These make up the central region of each of the facets of the icosahedron. Protein IX is believed to function as a capsid 'cement'. So far, its structure in not known. Although viruses lacking pIX do not form GONs and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains a putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. To get more insight into the structure/function relationship we studied the incorporation of heterologous pIX molecules into the HAdV-5 capsid. Our data show that the pIX proteins of HAdV-12 (species A) and HAdV-7 (species B) can be efficiently incorporated into the capsid of pIX-deleted HAdV-5 (species C). In addition, further deletion mutagenesis confirmed that the N-terminus of the HAdV-5 pIX is required and sufficient for incorporation into the capsid. These data further our knowledge on pIX and facilitated the development of pIX as an anchor for the incorporation of heterologous peptides into the capsid. In this way receptor-binding ligands were incorporated in the capsid. Inclusion of alphahelical spacers of up to 7.5 nm between the pIX anchor and the ligand resulted in more efficient binding of the virions to antibodies and cells. These data conform that modification of pIX can be used to incorporate heterologous peptides into the adenoviral capsid, and support the feasibility of this approach to amend the tissue tropism of the adenovirus vectors.

Although successful in many rodent models of malignant gliomas, results of clinical adenoviral gene therapy trials using replication deficient vectors have been disappointing. One of the explanations of the discrepancy between results in animal models and clinical trials is the low expression of the Coxsackie Adenovirus receptor (CAR), the main high affinity receptor for binding of adenovirus serotype 5, on primary glioma cells as opposed to established glioma cell lines. To obtain improved adenoviral vectors, we made use of the differential tropism of the naturally occurring 51 human adenoviral serotypes to test the hypothesis that adenoviral vectors, binding to other receptors than CAR, would result in improved infection of primary glioma cells.

A problem associated with the gene-therapy concept is the T cell-mediated immune response, which is elicited by synthesis and presentation of transgene-derived and vector-derived peptides and which leads to rapid clearance of the transduced cells and loss of transgene expression. One way to avoid the immune response is by blocking the ubiquitin/proteasome pathway. Interference in this pathway is widely applied by viruses, for instance by Epstein-Barr virus (EBV) in its nuclear antigen 1 (EBNA-1). EBNA-1 is required for the maintenance of the viral episomes in the infected cells. Although EBNA-1 specific CTL have been described, they cannot recognize EBV-infected cells. The failure to recognize endogenously expressed EBNA-1 has previously been attributed to a glycine-alanine rich (GAr) stretch in the EBNA-1 sequence that protects the protein from proteasomal degradation. We have shown previously that inclusion of the full length EBNA-1 derived GAr domain could be exploited to abrogate CTL-induced killing of cells expressing the transgene (ref 1). Firstly we demonstrated that inclusion of the EBNA-1 derived GAr domain does not interfere with enzyme function. We tested three different enzymes that all retained their activity. In addition, a recombinant adenovirus expressing GAr-containing β-galactosidase was able to deliver functional enzyme in vivo. However, expression of the GAr-β-gal as well as the control β-gal resulted in the induction of a strong β-galactosidase-specific CTL response. This can be attributed to cross-priming, as vaccination of BALB/c mice (H-2d) with C57Bl/6 (H-2b) fibroblasts expressing GAr-β-galactosidase resulted in the induction of β-gal-specific CTLs. Nonetheless, LacZ-specific CTL did not recognize target cells expressing GAr-LacZ in vitro. These results showed that the GAr can be exploited to create 'stealth' genes by hiding specific foreign antigens from CTL-mediated immune-attack. Currently, we are testing the hypothesis that fusion with the GAr will lead to a prolonged expression of transgenes in vivo. Preliminary evidence indicates that the modification leads to prolonged expression of β-gal in vivo after adenovirus-mediated intra-muscular gene transfer. In addition, we generated stable B16 mouse melanoma cell lines expressing either thymidine kinase (TK) or GAr-TK. The tumor take and survival of these cells in C57Bl/6 mice are being studied either in the presence or in the absence of antigen-specific CTL.

One important goal in gene therapy is to develop adenovirus (Ad) vectors that are genetically de-targeted as well as re-targeted. Genetic re-targeting of Ad using complex cell-binding ligands has previously not been possible. We have previously demonstrated that ligands for genetic re-targeting of adenoviruses must be able to fold correctly in the cytoplasm of virus producing cells, a milieu that is not conducive to the formation of disulphide bonds.

Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.

We have introduced insertion and deletion mutations in the cloned DNA binding protein (DBP) gene of adenovirus type 5. The mutated DBP genes were subsequently introduced in the viral genome by a combination of in vitro and in vivo methods. The resulting mutant viruses were tested for their viability in human 293 cells and an initial characterization of these viruses was performed. Viable mutants with insertions in the carboxyl-terminal portion of the gene could not be obtained. In contrast, a number of viable mutants were constructed that contained insertions or deletions in the amino-terminal half of DBP. Several of these, which covered the region between amino acid (aa) residues 39 and 81, were phenotypically wild type, implying that this segment is completely dispensable for DBP function. However, mutations altering the region encompassed by as 2–38 were, at least, partially defective suggesting that this region is important for full activity of the protein.

Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth.

Cultures of glial cells were prepared from the brains of one-week-old rat pups. After one day in culture, serum was omitted from the medium and replaced by a combination of growth-stimulating hormones and other factors that enhanced the percentage of oligodendrocytes in the cultures. We investigated the effects of hydrocortisone on the development of oligodendrocytes, on the activities of oligodendrocyte-specific enzymes and on glucose- and lipid-metabolism of the glial cells. (1) Hydrocortisone greatly enhanced the survival of glial cells in culture. (2) The development of galactocerebroside-positive cells and the specific activity of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase were stimulated by 50 nM hydrocortisone, whereas these effects were partly reversed at higher concentrations of the hormone. (3) The specific activity of glycerol-3-phosphate dehydrogenase was markedly stimulated by hydrocortisone; 1 μM or higher concentrations of hydrocortisone were required for an optimal effect. (4) The consumption of glucose and the production of lactate were lowered by hydrocortisone whereas the oxidation of [6-14C]glucose to 14CO2 was not affected. (5) Incorporation of [35S]sulfate into sulfolipids was greatly enhanced by hydrocortisone and [14C]incorporation from [1-14C]acetate into cholesterol and fatty acids was also stimulated but to a smaller extent. These results show that hydrocortisone (i) exerts a general trophic function on glial cells in our culture system; (ii) enhances the ratio of oligodendrocytes over astrocytes, possibly by directing bipotential progenitor cells to develop into oligodendrocytes; (iii) specifically induces glycerol-3-phosphate dehydrogenase in oligodendrocytes.