Pia Delaere

Direct intracerebral gene transfer of an adenoviral vector expressing tyrosine hydroxylase in a rat model of Parkinson's disease

DIRECT intracerebral gene transfer to neural cells has been demonstrated with recombinant adenovirus encoding β-galactosidase. To explore the potential of recombinant adenovirus for the therapy of neurological disease we constructed a recombinant adenovirus encoding tyrosine hydroxylase, and optimized intracerebral injection to express the gene in the striatum of unilaterally denervated rats. These animals have dopamine depletion in their lesioned striatum, causing a rotation asymmetry induced by apomorphine. One, and two weeks after intracerebral injection this sensorimotor asymmetry was decreased by the adenovirus encoding tyrosine hydroxylase, and not by a control adenovirus encoding β-galactosidase. Histological analysis showed that tyrosine hydroxylase was preferentially expressed in astrocytes.

Continuous delivery of neurotrophin 3 by gene therapy has a neuroprotective effect in experimental models of diabetic and acrylamide neuropathies.

Neurotrophic factors (NFs) are promising agents for the treatment of peripheral neuropathies such as diabetic neuropathy. However, the value of treatment with recombinant NF is limited by the short half-lives of these molecules, which reduces efficiency, and by their potential toxicity. We explored the use of intramuscular injection of a recombinant adenovirus encoding NT-3 (AdNT-3) to deliver sustained low doses of NT-3. We assessed its effect in two rat models: streptozotocin (STZ)-induced diabetes, a model of early diabetic neuropathy characterized by demyelination, and acrylamide experimental neuropathy, a model of diffuse axonal neuropathy which, like late-onset human diabetic neuropathy, results in a diffuse sensorimotor neuropathy with dysautonomy. Treatment of STZ-diabetic rats with AdNT-3 partially prevented the slowing of motor and sensory nerve conduction velocities (p < 0.01 and p < 0.0001, respectively). Treatment with AdNT-3 of acrylamide-intoxicated rats prevented the slowing of motor and nerve conduction velocities (p < 0.001 and p < 0.0001, respectively) and the decrease in amplitude of compound muscle potentials (p < 0.0001), an index of denervation. Acrylamide-intoxicated rats treated with NT-3 had higher than control levels of muscle choline acetyltransferase activity (p < 0.05), suggesting greater muscle innervation. In addition, treatment of acrylamide-intoxicated rats with AdNT-3 significantly improved behavioral test results. Treatment with AdNT-3 was well tolerated with minimal muscle inflammation and no detectable general side effects. Therefore, our results suggest that NT-3 delivery by adenovirus-based gene therapy is a promising strategy for the prevention of both early diabetic neuropathy and axonal neuropathies, especially late axonal diabetic neuropathy.

Proteolytical processing of mutated human amyloid precursor protein in transgenic mice.

The evidence that betaA4 is central to the pathology of Alzheimers disease (AD) came from the identification of several missense mutations in the amyloid precursor protein (APP) gene co-segregating with familial AD (FAD). In an attempt to study the proteolytical processing of mutated human APP in vivo, we have created transgenic mice expressing the human APP695 isoform with four FAD-linked mutations. Expression of the transgene was controlled by the promoter of the HMG-CR gene. Human APP is expressed in the brain of transgenic mice as shown by Western blot and immunohistology. The proteolytic processing of human APP in the transgenic mice leads to the generation of C-terminal APP fragments as well as to the release of betaA4. Despite substantial amounts of betaA4 detected in the brain of the transgenic mice, neither signs of Alzheimers disease-related pathology nor related behavioural deficits could be demonstrated.

Partial Prevention of Cisplatin-Induced Neuropathy by Electroporation-Mediated Nonviral Gene Transfer

Cisplatin-induced sensory peripheral neuropathy is the dose-limiting factor for cisplatin chemotherapy. We describe the preventive effect of NT-3 delivery, using direct gene transfer into muscle by in vivo electroporation in a mouse model of cisplatin-induced neuropathy. Cisplatin-induced neuropathy was produced by weekly injections of cisplatin (five injections). Two doses of plasmid DNA encoding murine NT-3 (pCMVNT-3) were tested (5 and 50 microg/animal/injection). Cisplatin-treated mice were given two intramuscular injections. The first injection of pCMVNT-3 was given 2 days before the first injection of cisplatin and the second injection 2 weeks later. Six weeks after the start of the experiment, measurement of NT-3 levels (ELISA) demonstrated significant levels both in muscle and plasma. We observed a smaller cisplatin-related increase in the latency of the sensory nerve action potential of the caudal nerve in pCMVNT-3-treated mice than in controls (p < 0.0001). Mean sensory distal latencies were not different between the 5- and 50- microg/animal/injection groups. Treatment with gene therapy induced only a slight muscle toxicity and no general side effects. Therefore, neurotrophic factor delivery by direct gene transfer into muscle by electroporation is of potential benefit in the prevention of cisplatin-induced neuropathy and of peripheral neuropathies in general.

Regional and cellular presenilin 2 (STM2) gene expression in the human brain

Presenilin 2 (STM2) is a recently cloned gene involved in some forms of early onset Alzheimers disease with autosomal dominant inheritance. Here we report the regional and cellular distribution of STM2 mRNA in the normal human central nervous system. Using in situ hybridization. STM2 gene expression was shown to be confined exclusively to neurones in the central nervous system. A high level of STM2 mRNA expression was observed in the cerebral cortex and the hippocampus, more particularly on pyramidal neurones of Ammons horn and granular neurones of the dentate gyrus. STM2 mRNA was also detected in Purkinje cells and granular cells of the cerebellum, and in neurones of the striatum and the nucleus basalis of Meynert. Taken together, these results suggest that the expression of STM2 mRNA is not restricted to the neuronal populations that are known to degenerate in Alzheimers disease.

App, Apoe, and Presenilin Transgenics

Both environmental and genetic factors are involved in Alzheimer’s Disease (AD) aetiology. Mutations in the amyloid precursor protein (APP) and in presenilin PS1 and PS2 genes cause early-onset forms of the disease while the apolipoprotein ApoEe4 allele is a risk factor for AD (reviewed in Selkoe, 1996; Hardy, 1997). Environmental factors such as trauma and inflammation have also been implicated in the pathology but the overall mechanism of the disease is poorly understood, hampering the development of therapeutic treatments. An animal model of the disease would be of great interest to both unravel the pathophysiological mechanism in vivo and to provide a model for testing of therapeutic approaches. Recently, large overexpression of mutated forms of APP in two transgenic mouse models has been shown to lead to amyloid plaque formation and behavioral deficits (Games et al., 1995; Hsiao et al., 1996). However, mutations in APP and PS’s proteins have also been recently shown to contribute to a similar pathological process, the increase in production of the long form of Aβ (Aβ 1-42, Selkoe, 1996, Duff e

Recombinant adenoviruses coding for brain-derived neurotrophic factor (BDNF)

Xal., 1996; Borchelt et al., 1996), possibly through a direct physical interaction (Weidemann et al, 1997). Therefore, rather than large overexpression, transgenic models based on a combination of the known genetic factors expressed at more physiological levels could potentially lead to a more suitable model of the disease. Towards that goal, we have generated several human mutant APP, and PS’s transgenic rodent lines and combined them by breeding both together and with ApoE-KO animals.