Effect of retinol dehydrogenase gene transfer in a novel rat model of Stargardt disease.

Dysfunction of the ATPase-binding Cassette Transporter protein (ABCA4) can lead to early onset macular degeneration, in particular to Stargardt disease. To enable translational research into this form of blindness, we evaluated the effect of Cas9-induced disruptions of the ABCA4 gene to potentially generate new transgenic rat models of the disease. We show that deletion of the short exon preceding the second nucleotide-binding domain is sufficient to drastically knock down protein levels and results in accumulation of retinoid dimers similar to that associated with Stargardt disease. Overexpression of the retinol dehydrogenase enzymes RDH8 and RDH12 can to a limited extent offset the increase in the bisretinoid levels in the Abca4Ex42-/ - KO rats possibly by restricting the time window in which retinal can dimerize before being reduced to retinol. However, in vivo imaging shows that overexpression of RDH8 can induce retinal degeneration. This may be due to the depletion in the outer segment of the cofactor NADPH, needed for RDH function. The translational potential of RDH therapy as well as other Stargardt disease therapies can be tested using the Abca4 knockdown rat model.

Long-term expression of melanopsin and channelrhodopsin causes no gross alterations in the dystrophic dog retina.

Several preclinical studies have investigated the potential of algal channelrhodopsin and human melanopsin as optogenetic tools for vision restoration. In the present study, we assessed the potentially deleterious effects of long-term expression of these optogenes on the diseased retina in a large animal model of retinal degeneration, the RPE65-deficient Briard dog model of Leber congenital amaurosis. Intravitreal injection of adeno-associated virus vectors expressing channelrhodopsin and melanopsin had no effect on retinal thickness over a 16-month period post injection. Our data support the safety of the optogenetic approach for the treatment of blindness.

In the rat liver, Adenoviral gene transfer efficiency is comparable to AAV.

Adenoviral (AdV) and Adenovirus-associated viral (AAV) vectors both are used for in vivo gene therapy of inherited liver disorders, such as Crigler-Najjar syndrome type 1. In a relevant animal model, the Gunn rat, both vectors efficiently correct the severe hyperbilirubinemia characteristic of this liver disorder. Although the clinical use of AAV is more advanced, as demonstrated by the successful phase 1 trial in hemophilia B patients, because of its large cloning capacity AdV remains an attractive option. A direct comparison of the efficacy of these two vectors in the liver in a relevant disease model has not been reported. Aim of this study was to compare the efficiency of clinically applicable doses of both vectors in the Gunn rat. AdV or scAAV (self-complimentary AAV) ferrying identical liver-specific expression cassettes of the therapeutic gene, UGT1A1, were injected into the tail vein. As the titration methods of these two vectors are very different, a comparison based on vector titers is not valid. Therefore, their efficacy was compared by determining the amount of vector genomes delivered to the liver required for therapeutic correction of serum bilirubin. Like AAV, the liver-specific first-generation AdV also provided sustained correction in this relevant disease model. UGT1A1 mRNA expression provided per genome was comparable for both vectors. Flanking the expression cassette in AdV with AAV-ITRs (inverted terminal repeats), increased UGT1A1 mRNA expression eightfold which resulted in a significant improvement of efficacy. Compared with AAV, less AdV genomes were needed for complete correction of hyperbilirubinemia.

No tumour-initiating risk associated with scAAV transduction in newborn rat liver.

Delivery of recombinant adeno-associated virus (rAAV) vectors to the newborn liver is followed by a rapid loss of episomal vector copies because of hepatocyte proliferation. In selected hepatocytes, integration of rAAV genomes can lead to a sustained expression of the transgene. The safety of in vivo gene therapy with single-stranded AAV vectors has been questioned in a study reporting a high incidence of hepatocellular carcinoma, associated with provirus integration events in mice that receive an single-stranded AAV injection at birth. To investigate the tumour-initiating potential of the newly established self-complementary AAV (scAAV) vectors in the liver, groups of newborn rats received intravenous injection of a scAAV vector encoding the green fluorescent protein (GFP), or were injected with phosphate-buffered saline (PBS) or diethylnitrosamine (DEN), a well-known liver tumour initiator. The rats were fed on a diet containing 2-acetylaminofluorene, a potent liver tumour-promoting agent to accelerate the carcinogenic process. After 2 months, the animals were killed and their livers analysed. Preneoplastic nodules were identified by glutathion S-transferase-p (GSTp) staining, and GFP expression was detected by immunohistochemistry. Vector genome integration events were analysed. The numbers of GSTp-positive foci were comparable in the PBS and the scAAV-GFP groups and significantly higher in the DEN group. The proportion of GSTp-positive foci that also expressed GFP was low and in the range expected for random occurrence. No specific integration hot spots were detected by linear amplification-mediated-PCR in transduced liver. In conclusion, scAAV transduction of newborn rat liver does not trigger preneoplastic lesions suggesting an absence of liver tumourigenesis.

Critical assessment of lifelong phenotype correction in hyperbilirubinemic Gunn rats after retroviral mediated gene transfer.

Among inherited diseases of the liver, Crigler-Najjar type 1 disease (CN-1), which results from complete deficiency in bilirubin UDP-glucuronosyltransferase activity (B-UGT1), is an attractive target for gene therapy studies. Hyperbilirubinemic Gunn rats, a model of CN-1, were injected at 2 days of age with lentiviral or oncoretroviral vectors encoding the human B-UGT1. After injection, bilirubinemia was normalized for up to 95 weeks. Bilirubin conjugates were present in the bile, demonstrating liver transduction. PCR and enzyme activity analysis confirmed gene and phenotype correction in liver. We observed that when using a strong viral promoter, a complete correction was achieved with less than 5% of B-UGT1 copy per haploid genome and after a reconstitution of 12% B-UGT1 normal activity. Liver histology remained normal throughout the experiment and tissue distribution analysis revealed preferential hepatocyte transduction after systemic delivery. Finally, no adverse immune response occurred even after induction of nonspecific liver inflammation, suggesting immune ignorance to the therapeutic protein. Our present results document the lifelong safety of gene therapy for CN-1 with retroviral vectors. They offer a better delineation of liver gene correction level required to achieve complete correction of bilirubinemia and pave the way for future clinical application of gene therapy for inherited liver disorders.

Efficient retroviral gene transfer to the liver in vivo using nonpolypeptidic mitogens.

Recombinant retroviral vectors are attractive tools for achieving sustained expression of a therapeutic gene in the liver. However, cell division is required for efficient transduction with these vectors. Here we report that two widely used liver mitogens, triiodothyronin (T3) and cyproterone acetate (CPA), enable hepatocyte transduction with recombinant retroviral vectors delivered in vivo into the bloodstream. Treatment with T3 as well as CPA, alone or in combination, resulted in an increase in hepatocyte replication predominantly around the portal tract. The mitogenic activity made it possible to transduce hepatocytes in the same location. Moreover, when administered together, the two drugs synergized and the transduction level reached 5% of hepatocytes. This transduction level is compatible with clinical applications for a number of inherited liver diseases. Since these two compounds have a long history of safe clinical use, we propose that these liver mitogens may have potential for clinical application in liver-directed gene therapy.

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line.

We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. alpha3beta1, alpha5beta1, alpha6beta1/beta4, alpha8beta1 and alpha(v)beta1/beta5/beta6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, alpha2beta1, alpha(v)beta6 and alpha6beta1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas alpha(v)beta5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.

Adhesion, actin cytoskeleton organisation and the spreading of colon adenocarcinoma cells induced by EGF are mediated by alpha2beta1 integrin low clustering through focal adhesion kinase.

Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.

Effect of microtubule disruption on cell adhesion and spreading.

Microtubules have been involved in a variety of cellular processes. In this study, we examined the role of the microtubular system in the adhesion and spreading of the adenocarcinoma cell line HT29-D4. Disruption of microtubules by nocodazole or navelbine resulted in an increase in cell adhesion to purified ECM proteins. This enhanced cell adhesion is mediated by integrins, but is not attributable to quantitative changes in the number of integrin receptors at the cell surface, as determined by flow cytometric analysis. In contrast to attachment, spreading of HT29-D4 cells was reduced by nocodazole treatment in a dose-dependent manner. Thus, microtubule depolymerization appears to increase initial attachment of cells to extracellular matrix, while impeding subsequent cell spreading.

Convergent effects of growth factors, hormones, and fibronectin are necessary for the enterocyte differentiation of a colon adenocarcinoma cell line (HT29-D4).

The aim of this work was to show in serum-free medium a convergent effect of physiological factors and extracellular matrix proteins on the differentiation process of enterocytes by taking as a model the HT29-D4 clone that has the feature of differentiating when subcultured in fetal bovine serum glucose-free medium. We show that triiodothyronine (T3) as well as insulin promotes limited cell growth and differentiation, whereas fibronectin or bovine serum albumin (BSA) induces cell growth and a low level of differentiation. However, insulin, T3, fibronectin, and BSA together with epidermal growth factor and transferrin promoted satisfactory growth and enterocyte morphology with epithelial electrophysiological properties in HT29-D4 cells. With these factors adequate protein targeting was achieved since cells apically expressed the carcinoembryonic antigen, and basolaterally transferrin and insulin receptors, beta 1 and alpha v beta 6 integrins, talin, vinculin, and focal adhesion kinase (FAK). Talin, vinculin, FAK, and alpha v beta 6 integrin, the fibronectin receptor, were clustered in focal contacts, which agrees with a possible role of fibronectin in final cell growth, the latter process mediating the final phase of differentiation. This level of differentiation can be maintained for a long time. Thus HT29-D4 cells appear to be a suitable model to study the implication of integrins in the differentiation process of human enterocytes.