Convection Enhanced Delivery of the Oncolytic Adenovirus Delta24-RGD in Patients with Recurrent GBM: A Phase I Clinical Trial Including Correlative Studies.

PURPOSE: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma. PATIENTS AND METHODS: Dose-escalation phase I study with 3+3 cohorts, dosing 107 to 1 × 1011 viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding. RESULTS: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2-associated cytokine levels and numbers of CD3+ T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+ and CD8+ T cells. No evidence of viral shedding in excreta was observed. CONCLUSIONS: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses.

Audiogenic seizure susceptibility is reduced in fragile X knockout mice after introduction of FMR1 transgenes.

The Fmr1 knockout (KO) mouse is characterized by an increased audiogenic seizure (AGS) susceptibility and is considered a good animal model for epilepsy and seizures in the human fragile-X (FRAX) syndrome. Here, we tested the hypothesis that the reintroduction of the FMR1 gene is able to revert the AGS susceptibility characterizing Fmr1 KO mice. To this aim, two groups of Fmr1 KO transgenic mice, which have additional copies of the human FMR1 gene (YAC) or FMR1 cDNA (G6) were used. AGS susceptibility of these mice was examined and compared to that of Fmr1 KO, wild type, and wild-type animals in whom the FMR1gene was also introduced (over-expressed). Mice were tested at different ages because AGS susceptibility is age dependent. The intensity of response was scored and the results were analyzed by means of 2-way analysis of variance to evaluate the effects of age and genetic condition. We found that AGS susceptibility rescue is complete in the G6 mice and partial in YAC mice. Our data indicate that the introduction of the human FMR1 gene in Fmr1 KO mice is able to revert the Fmr1 KO epileptic phenotype.

Altered differentiation of neural stem cells in fragile X syndrome.

Fragile X syndrome, a common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a mutation in the FMR1 gene. We investigated the differentiation of neural stem cells generated from the brains of fmr1-knockout (KO) mice and from postmortem tissue of a fragile X fetus. Mouse and human FMRP-deficient neurospheres generated more TuJ1-positive cells (3-fold and 5-fold, respectively) than the control neurospheres generated from normal mouse and human brains, and these cells showed morphological alterations with fewer and shorter neurites and a smaller cell body volume. The number of cells expressing glial fibrillary acidic protein and generated by these neurospheres was reduced because of increased apoptotic cell death. Furthermore, there was an increase in a population of cells with intense oscillatory Ca2+ responses to neurotransmitters in differentiated cells lacking FMRP. In addition, the number of cells in a cohort of bromodeoxyuridine-labeled newborn cells was increased in the subventricular zone of the telencephalon of the fmr1-KO mouse in vivo. These results demonstrate substantial alterations in the early maturation of FMRP-deficient neural stem cells in fragile X syndrome and in the fmr1-KO mice.

BDNF regulates the expression of fragile X mental retardation protein mRNA in the hippocampus.

Both fragile X mental retardation protein (FMRP) and brain-derived neurotrophic factor (BDNF) are implicated in the maturation of neurons and in the higher cognitive functions. We have investigated whether FMRP and BDNF are reciprocally regulated in neurons. Exposure of cultured hippocampal neurons to BDNF, but not to NT-3, reduced FMR1 mRNA levels to 84.8% of control at 4 h and the levels were back to baseline by 24 h or 4 days. Furthermore, expression of FMR1 mRNA was reduced (82.4% of control) in vivo in the hippocampus of transgenic mice overexpressing TrkB receptors, and a small but significant (5.1%) decrease was also detected in FMRP protein levels. In contrast, the expression patterns of BDNF and TrkB mRNAs were not altered in FMRP-deficient mice compared to wild-type mice. Our data provide evidence that BDNF via TrkB signaling decreases FMRP expression and suggest a role for FMRP in BDNF-induced synaptic plasticity.

Knockout mouse model for Fxr2: a model for mental retardation.

Fragile X syndrome is a common form of mental retardation caused by the absence of the FMR1 protein, FMRP. Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macro-orchidism and behavioral abnormalities. Two homologs of FMRP have been identified, FXR1P and FXR2P. These proteins show high sequence similarity, including all functional domains identified in FMRP, such as RNA binding domains. They have an overlap in tissue distribution to that of FMRP. Interactions between the three FXR proteins have also been described. FXR2P shows high expression in brain and testis, like FMRP. To study the function of FXR2P, we generated an Fxr2 knockout mouse model. No pathological differences between knockout and wild-type mice were found in brain or testis. Given the behavioral phenotype in fragile X patients and the phenotype previously reported for the Fmr1 knockout mouse, we performed a thorough evaluation of the Fxr2 knockout phenotype using a behavioral test battery. Fxr2 knockout mice were hyperactive (i.e. traveled a greater distance, spent more time moving and moved faster) in the open-field test, impaired on the rotarod test, had reduced levels of prepulse inhibition, displayed less contextual conditioned fear, impaired at locating the hidden platform in the Morris water task and were less sensitive to a heat stimulus. Interestingly, there are some behavioral phenotypes in Fxr2 knockout mice which are similar to those observed in Fmr1 knockout mice, but there are also some different behavioral abnormalities that are only observed in the Fxr2 mutant mice. The findings implicate a role for Fxr2 in central nervous system function.