Search

Adenovirus-mediated transfer of the nerve growth factor gene promotes significant recovery of age-related cholinergic neuronal deficits in aged rats, but the effects of such treatment on cognitive dysfunction remain unclear. Herein we report a beneficial effect of first-generation adenovirus-mediated nerve growth factor gene transfer (AdNGF) on the spatial learning and memory of aged rats. The NGF protein was detected by enzyme-linked immunosorbent assay in cerebrospinal fluid as early as 3 days after gene transfer and was expressed for at least 30 days. Escape latency in the Morris water maze hidden-platform test was significantly improved on day 8 postinoculation in memory-impaired rats treated with AdNGF as well as at later testing intervals. Ultimately, the escape latency values for the AdNGF group become indistinguishable from those for aged rats with normal learning capacity. Immunohistochemical analysis of septal cholinergic neurons for choline acetyltransferase (ChAT) showed significant increases in both the number and somal distribution of ChAT-positive cells after inoculation of memory-impaired rats with AdNGF. Improvement in memory performance was positively correlated with increases in both NGF concentration in cerebrospinal fluid (r = 0.73, p = 0.005) and the number of ChAT-staining cells (r = 0.77, p = 0.0022). We conclude that AdNGF can improve cognitive function in memory-impaired aged rats and, with refinements in vector-driven expression of the transgene, may prove suitable for use in humans.

Between December 1996 and September 1998, 13 patients with advanced recurrent malignant brain tumors (9 with glioblastoma multiforme, 1 with gliosarcoma, and 3 with anaplastic astrocytoma) were treated with a single intratumoral injection of 2 × 109, 2 × 1010, 2 × 1011, or 2 × 1012vector particles (VP) of a replication-defective adenoviral vector bearing the herpes simplex virus thymidine kinase gene driven by the Rous sarcoma virus promoter (Adv.RSVtk), followed by ganciclovir (GCV) treatment. The VP to infectious unit ratio was 20:1. Our primary objective was to determine the safety of this treatment. Injection of Adv.RSVtk in doses ≤2 × 1011VP, followed by GCV, was safely tolerated. Patients treated with the highest dose, 2 × 1012VP, exhibited central nervous system toxicity with confusion, hyponatremia, and seizures. One patient is living and stable 29.2 months after treatment. Two patients survived >25 months before succumbing to tumor progression. Ten patients died within 10 months of treatment, 9 from tumor progression and 1 with sepsis and endocarditis. Neuropathologic examination of postmortem tissue demonstrated cavitation at the injection site, intratumoral foci of coagulative necrosis, and variable infiltration of the residual tumor with macrophages and lymphocytes.Molecular Therapy (2000) 1, 195–203; doi: 10.1006/mthe.2000.0030

Schwann cells in the intact sciatic nerve express high amounts of ciliary neurotrophic factor (CNTF), but 7 days after injury to the nerve expression dramatically decreases. To determine whether this change occurs only in the region of the injury or throughout the whole nerve we examined the spatial and temporal expression of CNTF after a crush injury. One day after injury the amount of CNTF mRNA and protein decreased within the first 4 mm distal to the crush site. This decrease progressed in a centrifugal manner distally until mRNA and protein were scarcely detectable by 7 days. In nerve proximal to the crush site CNTF expression decreased slightly and was still detectable at all sample times. During regeneration CNTF expression remained very low up to 14 days after injury. By 30 days mRNA and protein were detectable and by 60 days CNTF protein was present at normal amounts. Immunohistochemical analysis of normal nerve revealed CNTF localized in outer portion of the cytoplasm of myelin-forming Schwann cells. Three days after injury CNTF coalesced with pockets of cytoplasm in the Schwann cell and by 5 days was barely detectable. Positive staining remained in proximal segments where little or no degeneration occurred. These results demonstrate that CNTF expression in Schwann cells is synchronized with their functional state. CNTF expression decreases with demyelination during Wallerian degeneration and returns to normal following remyelination during regeneration. These findings also suggest that CNTF expression requires intact axon-Schwann cell interactions. Copyright 1993, 1999 Academic Press

ABSTRACTWe investigated the therapeutic efficacy of adenovirus-mediated gene therapy to treat malignant mammary tumors in vitroand in vivoin the brain. A mammary adenocarcinoma cell line derived from Fischer rats (13762 MAT B III; MAT-B) was used. In vitrostudies demonstrated that the MAT-B cells could be efficiently transduced with a replication-defective adenovirus (ADV) vector that carried the herpes simplex virus gene for thymidine kinase (ADV-tk), and that ADV-tk transduction rendered the MAT-B cells sensitive to killing, in a dose-dependent manner, with ganciclovir (GCV). An animal model of a mammary tumor metastatic to the brain was produced by injecting MAT-B cells into the caudate nucleus of Fischer rats. Seven days after MAT-B cell injection, when the tumors were approximately 5 mm2in cross-sectional size, the tumors were injected with ADV-tk or a control adenovirus vector containing the -galactosidase (-Gal) gene (ADV-gal). After vector injection the animals were treated with GCV or with saline for 6 days. Sixteen days after tumor cell injection, the brains were examined histologically. The rats that were injected with ADV-gal and treated with GCV or saline, and those that were injected with ADV-tk and treated with saline had large tumors, whereas the rats that were injected with ADV-tk and treated with GCV had no visible tumor tissue at the site of tumor cell injection. In survival studies animals treated with ADV-tk GCV survived a significantly longer time than animals treated with ADV-gal GCV. Our results demonstrate that the recombinant adenoviral vector containing the tkgene confers GCV cytotoxic sensitivity to mammary tumor cells in vitroand in the brain, and suggest that this treatment strategy may be useful in treating somatic tumors that metastasize to the brain.

Shiverer (shi) is an autosomal recessive mutation in mice that results in hypomyelination in the central nervous system (CNS) but normal myelination in the peripheral nervous system (PNS). Myelin basic proteins (MBPs) are virtually absent in both PNS and CNS. It is not known whether the cellular target in the PNS is the myelin-forming Schwann cell or another cell type which secondarily affects the Schwann cell. To determine the cellular target of the shi gene, we have adapted tissue culture techniques that allow co-culture of pure populations of mouse sensory neurons of one genotype with Schwann cells and fibroblasts of another genotype under conditions that permit myelin formation. These cultures were stained immunocytochemically as whole mounts to determine whether MBPs were expressed under various in vitro conditions. In single-genotype cultures, presence or absence of MBPs was consistent with earlier in vivo results: +/+ cultures were MBP-positive and shi/shi cultures were MBP-negative. In mixed-genotype cultures, visualization of MBPs in myelin accorded with the genotype of the non-neuronal Schwann cells and fibroblasts and not with the neurons--those cultures that contained +/+ non-neuronal cells were MBP-positive and those with shi/shi non-neuronal cells were MBP-negative, independent of the neuronal genotype. These results rule out neurons or circulating substances as mediators of the influence of the shi genetic locus on MBP synthesis and deposition in peripheral myelin.