Adrian E. Morelli

Treatment of Experimental Glioma by Administration of Adenoviral Vectors Expressing Fas Ligand

Fas ligand (FasL) is a cytokine, produced by activated T cells and NK cells, that triggers apoptosis of Fas-positive target cells including human glioma cells. As shown here, in vitro infection of rat F98 and human LN18 glioma cell lines with recombinant adenovirus (rAd) expressing FasL cDNA under control of the cytomegalovirus promoter (rAd-CMV-FasL) induced striking cytotoxicity in Fas-positive glioma cell lines but not in the Fas-negative F98 glioma subline F98/ZH. The extent of FasL-mediated cytotoxic effects outranged the expectations based on expression of beta-galactosidase (beta-Gal) by F98 cells infected with a control virus expressing the lacZ gene (rAd-CMV-lacZ). The detection of FasL bioactivity in supernatants of infected cells provides evidence of a bystander mechanism involving the cytotoxic action of FasL on uninfected cells. In F98 tumor-bearing rats, infection with rAd-CMV-FasL increased the mean survival time by 50% compared with infection with rAd-CMV-lacZ or untreated controls. These data suggest that viral vector transduction of the FasL gene could be part of a successful glioma gene therapy.

Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK.

Herpes simplex virus type 1-thymidine kinase (HSV1-TK) in combination with ganciclovir is an efficient and widely used strategy in brain tumour gene therapy. Recently, we have shown effective inhibition of glioma growth in a syngeneic rat model using recombinant adenoviruses expressing the full-length HSV1-TK and an N-terminus truncated variant, HSV1-ΔTK in the presence of ganciclovir. We also showed active chronic brain inflammation in the long-term survivors (3 months) treated with HSV1-TK plus GCV. Furthermore, our results indicated loss of myelinated fibres, oedema and indices of ongoing axonal degeneration. In this study, we assessed the cytotoxicity of both HSV1-TK variants in the presence or absence of ganciclovir, in primary cultures of neurones and glia, and in the rat brain in vivo. Our results indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotoxicity for either neurones or glial cells grown in primary cultures, (2) on its own the full-length HSV1-TK is more cytotoxic than its truncated version HSV1-ΔTK for a population of non-neuronal and non-glial cells within neocortical primary cultures, and (3) in vivo, when delivered into the striatum, RAds encoding HSV1-TK are more cytotoxic than RAds encoding HSV1-ΔTK, after administration of ganciclovir. The effectiveness of HSV1-ΔTK in preventing brain tumour growth in vivo, combined with its reduced cytotoxicity, both in vivo and in primary cultures of CNS cells, could represent an advantage for treatment of brain tumours using gene therapy.

FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors.

Human embryonic kidney 293 cells contain the E1 region of adenovirus type 5, and thus sustain, through transcomplementation, the production of recombinant E1-deleted adenovirus vectors. During attempts to produce recombinant adenovirus expressing the apoptosis-inducing molecule Fas ligand (FasL) under the control of a very strong truncated major immediate–early human cytomegalovirus (MIEhCMV) promoter, we discovered that 293 cells were not surviving the initial cotransfection with a shuttle plasmid encoding the mouse FasL; and pJM17, a plasmid containing the genome of adenovirus type 5 with deletions in the E1-E3 regions, in an unpackagable form. Investigation of the reason for massive cell death after cotransfection led us to determine that 293 cells express the FasL receptor, Fas-Apo1 (CD95), and respond with apoptosis to the cross-linking of Fas-Apo1 with either IgM monoclonal antibodies or FasL. Therefore, we decided to generate adenoviral vectors expressing FasL under the control of tissue-specific and/or -inducible promoter elements. Our findings can explain difficulties several groups have had in generating recombinant adenoviral vectors expressing FasL using 293 cells, as well as the lower titres reported.

Uptake of α-(L)-iduronidase produced by retrovirally transduced fibroblasts into neuronal and glial cells in vitro

The uptake of recombinant α-(L)-iduronidase into glial and neuronal cells, produced by retrovirally transduced NIH3T3 fibroblasts, was studied. We demonstrate that: (1)u2009neuronal and glial cells take up α-(L)-iduronidase released into the medium by retrovirally transduced fibroblasts expressing high levels of α-(L)-iduronidase; (2)u2009both glial and neuronal cells express the cation independent mannose-6-phosphate receptor responsible for lysosomal enzyme uptake; and (3)u2009uptake of the lysosomal enzyme can be blocked by excess free mannose-6-phosphate, but not glucose-6-phosphate. Thus, various brain cells take up α-(L)-iduronidase, possibly through a cation independent mannose-6-phosphate receptor mediated pathway, and this uptake is higher in actively dividing or immature brain cells. Consequently, (1)u2009neuronal metabolism ought to be capable of cross correction by enzyme provided by genetically engineered and transplanted cells provided by bone marrow transplantation (BMT); (2)u2009that BMT could have a more beneficial effect on neurological function if performed as early as possible; and (3)u2009given that the uptake mechanism of glial cells has a higher capacity, it might be easier to target diseases like the leukodystrophies in which lysosomal enzymes are needed in glial cells, compared to diseases where lysosomal enzymes ought to be delivered into neurons.

Adenoviruses encoding HPRT correct biochemical abnormalities of HPRT- deficient cells and allow their survival in negative selection medium

The Lesch-Nyhan syndrome is an X-linked disorder caused by a virtually complete absence of the key enzyme of purine recycling, hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is characterized by uric acid overproduction and severe neurological dysfunction. No treatment is yet available for the latter symptoms. A possible long-term solution is gene therapy, and recombinant adenoviruses have been proposed as vectors for gene transfer into postmitotic neuronal cells. We have constructed an adenoviral vector expressing the human HPRT cDNA under the transcriptional control of a short human cytomegalovirus major immediate early promoter (RAd-HPRT). Here we show that infection of human 1306, HPRT-negative cells with RAd-HPRT, expressed high enough levels of HPRT enzyme activity, as to reverse their abnormal biochemical phenotype, thus enhancing hypoxanthine incorporation and restoring purine recycling, increasing GTP levels, decreasing adenine incorporation, and allowing cell survival in HAT medium in which only cells expressing high levels of HPRT can survive. Infection of murine STO cells, increased hypoxanthine incorporation and restored purine recycling, thus allowing cell survival in HAT medium, and reduced de novo purine synthesis. Although both cells were able to survive in HAT medium post infection with RAd-HPRT, some of the biochemical consequences differed. In summary, even though adenoviral vectors do not integrate into the genome of target HPRT-deficient human or murine cells, RAd-HPRT mediated enzyme replacement corrects abnormal purine metabolism, increases intracellular GTP levels, and allows cells to survive in a negative selection medium.

Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors

We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-βgal) containing the lacZ gene encoding β-galactosidase (β-gal), under the transcriptional control of the HSV1 immediate–early 3 (IE3) promoter; (2) RAd35, an E1−/E3− replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1−/E3− replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected β-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and β-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.

Adenoviruses Encoding HPRT Correct the Biochemical Abnormalities Fully Only in HPRT-Deficient Human Cell Lines: Importance of Species Differences

The Lesch-Nyhan syndrome (LNS) is an X-linked disorder caused by a virtually complete absence of the key purine recycling enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT) It is characterised by uric acid overproduction and severe neurological dysfunction. Despite the early discovery of the link between the neurological symptoms and a deficit in purine salvage the cellular and molecular mechanisms leading to LNS remain unexplained1. Various HPRT deficient ‘knock-out’ mouse models have shown the gross purine overproduction of LNS patients, but not the neurological abnormalities2. An interesting aspect of LNS is that it lacks progressive neurodegeneration. As yet there is no effective treatment. A possible solution is gene therapy, and recombinant adenoviruses have been proposed as vectors for gene transfer into postmitotic neuronal cells3.