ABSTRACT
Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by a mutation in the X-linked CDKL5 gene. CDKL5 is a serine/threonine kinase that is critical for axon outgrowth and dendritic morphogenesis as well as synapse formation, maturation, and maintenance. This disorder is characterized by early-onset epilepsy, hypotonia, and failure to reach cognitive and motor developmental milestones. Because the disease is monogenic, delivery of the CDKL5 gene to the brain of patients should provide clinical benefit. To this end, we designed a gene therapy vector, adeno-associated virus (AAV)9.Syn.hCDKL5, in which human CDKL5 gene expression is driven by the synapsin promoter. In biodistribution studies conducted in mice, intracerebroventricular (i.c.v.) injection resulted in broader, more optimal biodistribution than did intra-cisterna magna (i.c.m.) delivery. AAV9.Syn.hCDKL5 treatment increased phosphorylation of EB2, a bona fide CDKL5 substrate, demonstrating biological activity in vivo. Our data provide proof of concept that i.c.v. delivery of AAV9.Syn.hCDKL5 to neonatal male Cdkl5 knockout mice reduces pathology and reduces aberrant behavior. Functional improvements were seen at doses of 3e11 to 5e11 vector genomes/g brain, which resulted in transfection of ≥50% of the neurons. Functional improvements were not seen at lower doses, suggesting a requirement for broad distribution for efficacy.
ABSTRACT
Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors.
Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/pathology , Glioma/pathology , Histones/genetics , Mutation , Adolescent , Adult , Animals , Autopsy , Brain Neoplasms/genetics , Child , Child, Preschool , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioma/genetics , Humans , Infant , Male , Mice, Inbred NOD , Mice, SCID , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Young AdultABSTRACT
Corticosteroids, such as dexamethasone, are routinely used as palliative care in neuro-oncology for their anti-inflammatory benefits, however many patients experience dose limiting side effects caused by glucocorticoid response element (GRE)-mediated transcription. The purpose of this study was to use a murine model to investigate a new steroid alternative, vamorolone, which promises to reduce side effects through dissociating GRE-mediated transcription and NF-κB -mediated anti-inflammatory actions. To compare vamorolone to dexamethasone in reducing pro-inflammatory signals in vitro, murine glioma cells were treated with dexamethasone, vamorolone or vehicle control. Changes in mRNA expression were assessed using the nanostring inflammatory platform. Furthermore, drug efficacy, post-treatment behavioral activity and side effects were assessed by treating two cohorts of brain tumor bearing mice with dexamethasone, vamorolone, or vehicle control. Our investigation showed that treatment with vamorolone resulted in a reduction of pro-inflammatory signals in tumor cells in vitro similar to treatment with dexamethasone. Treatment with vamorolone resulted in a better safety profile in comparison to dexamethasone treatment. Vamorolone- treated mice showed similar or better activity and survival when compared to dexamethasone-treated mice. Our data indicate vamorolone is a potential steroid-sparing alternative for treating patients with brain tumors.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Brain Neoplasms/drug therapy , Cytokines/metabolism , Dexamethasone/pharmacology , Glioma/drug therapy , Inflammation Mediators/metabolism , Steroids/pharmacology , Animals , Anti-Inflammatory Agents/toxicity , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Cytokines/genetics , Dexamethasone/toxicity , Down-Regulation , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/metabolism , Glioma/pathology , Mice, Inbred NOD , Mice, SCID , Motor Activity/drug effects , NF-kappa B/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effectsABSTRACT
The objective of this study was to evaluate the effect of chemical tissue bonding (CTB) on adhesion strength, fluid permeability, and cell viability across a cartilaginous graft-host interface in an in vitro autologous chondral transplant (ACT) model. Chitosan-based cross-linkers; Chitosan-Rose Bengal [Chi-RB (Ch-ABC)], Chitosan-Genipin [Chi-GP (Ch-ABC)], and Chitosan-Rose Bengal-Genipin [Chi-RB-GP (Ch-ABC)] were applied to bovine immature cartilage explants after pre-treatment with surface degrading enzyme, Chondroitinase-ABC (Ch-ABC). Adhesion strength, fluid permeability and cell viability were assessed via mechanical push-out shear testing, fluid transport and live/dead cell staining, respectively. All three chitosan-based cross-linkers significantly increased the adhesion strength at the graft-host interface, however, only a statistically significant decrease in fluid permeability was noted in Chi-GP (Ch-ABC) specimen compared to untreated controls. Cell viability was maintained for 7 days of culture across all three treatment groups. These results show the potential clinical relevance of novel chitosan-based hydrogels in enhancing tissue integration and reducing synovial fluid penetration after ACT procedures in diarthoidal joints such as the knee and ankle. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1139-1146, 2016.
Subject(s)
Biocompatible Materials/administration & dosage , Cartilage/transplantation , Chitosan/administration & dosage , Animals , Cattle , Cell Survival , Female , Hydrogels , Male , Transplantation, AutologousABSTRACT
Surface damage to articular cartilage is recognized as the initial underlying process causing the loss of mechanical function in early-stage osteoarthritis. In this study, we developed structure-modifying treatments to potentially prevent, stabilize or reverse the loss in mechanical function. Various polymers (chondroitin sulfate, carboxymethylcellulose, sodium hyaluronate) and photoinitiators (riboflavin, irgacure 2959) were applied to the surface of collagenase-degraded cartilage and crosslinked in situ using UV light irradiation. While matrix permeability and deformation significantly increased following collagenase-induced degradation of the superficial zone, resurfacing using tyramine-substituted sodium hyaluronate and riboflavin decreased both values to a level comparable to that of intact cartilage. Repetitive loading of resurfaced cartilage showed minimal variation in the mechanical response over a 7 day period. Cartilage resurfaced using a low concentration of riboflavin had viable cells in all zones while a higher concentration resulted in a thin layer of cell death in the uppermost superficial zone. Our approach to repair surface damage initiates a new therapeutic advance in the treatment of injured articular cartilage with potential benefits that include enhanced mechanical properties, reduced susceptibility to enzymatic degradation and reduced adhesion of macrophages.