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ABSTRACT: It has been postulated that brain tumor stem cells (TSCs) may be the population of cells responsible for the maintenance and recurrence of glioblastoma multiforme (GBM). The purpose of this study was to optimize a reproducible protocol for generating TSCs for their subsequent transfection or transduction. Patient GBMs were enzymatically and mechanically dissociated and tumor spheres were resuspended in appropriate media and analyzed to ensure they met stem cell criteria. These cells were then transfected with a plasmid or transduced with a viral vector to introduce a previously absent gene and then allowed to form tumor spheres. Tumor spheres were generated from patient GBMs without contamination. These cells met stringent criteria as stem cells, including multipotentiality and self-renewal. High efficiency transfection and transduction of tumor spheres was possible, even at the core of the sphere. This allowed for the introduction of new genes to the TSCs, as evidenced by fluorescent microscopy and Western blot analysis. This study is a guide to optimize the generation of patient derived GBM tumor spheres without RBC and dead cell contamination. GBM TSCs within tumor spheres can easily be transfected with plasmids or transduced with a virus. This is important from a therapeutic perspective if gene replacement is to be successful in replacing genes lost in GBM progression or to knock down or silence genes that are over-expressed in malignant brain tumors.
Journal of Neuro-Oncology 02/2011; 104(2):509-22. · 3.21 Impact Factor
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ABSTRACT: Improvement in gait after shunt placement has been well documented in idiopathic normal pressure hydrocephalus (iNPH); however, controversy remains regarding the extent and pattern of postsurgical cognitive changes. Conflicting findings may be explained by variability in both test selection and follow-up intervals across studies. Furthermore, most investigations lack a control group, making it difficult to disentangle practice effects from a true treatment effect.
To examine postshunt changes in a sample of well-characterized iNPH participants compared with a group of age- and education-matched healthy control subjects.
We identified 12 participants with iNPH undergoing shunt placement and 9 control participants. All participants were evaluated with comprehensive neuropsychological testing and standardized gait assessment at baseline and were followed up for 6 months.
Repeated-measures analysis of variance revealed a significant group- (iNPH and control) by-time (baseline and 6 months) interaction for Trailmaking Test B: (P < .003) and Symbol Digit Modalities (P < .02), with greater improvement in iNPH participants relative to control subjects. In addition, the iNPH group showed greater improvement in gait (P < .001) and caregivers reported improved activities of daily living (P < .01) and reduced caregiver distress (P < .01).
This study demonstrates improvements in mental tracking speed and sustained attention 6 months after shunt placement in iNPH. The present investigation is the first study to use a controlled design to show that cognitive improvement in iNPH is independent of practice effects. Furthermore, these findings indicate functional and quality-of-life improvements for both the shunt responder and their caregiver.
Neurosurgery 11/2010; 68(2):416-9. · 2.79 Impact Factor
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Ronald G Crystal,
Dolan Sondhi,
Neil R Hackett,
Stephen M Kaminsky,
Stefan Worgall,
Philip Stieg,
Mark Souweidane,
Syed Hosain,
Linda Heier,
Douglas Ballon,
Miles Dinner,
Krystyna Wisniewski, Michael Kaplitt,
Bruce M Greenwald,
Joy D Howell,
Kristin Strybing,
Jonathan Dyke,
Henning Voss
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ABSTRACT: Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal childhood neurodegenerative lysosomal storage disease with no known therapy. There are estimated to be 200 to 300 children in the United States at any one time with the disease. LINCL is a genetic disease resulting from a deficiency of tripeptidyl peptidase I (TPP-I), a proteolytic enzyme encoded by CLN2, the gene that is mutated in individuals with LINCL. The subjects are chronically ill, with a progressive CNS disorder that invariably results in death, typically by age 8 to 12 years. The strategy of this clinical study is based on the concept that persistent expression in the CNS of the normal CLN2 cDNA with production of sufficient amounts of TPP-I should prevent further loss of neurons, and hence limit disease progression. To assess this concept, an adeno-associated virus vector (AAV2CUh-CLN2) will be used to transfer to and express the human CLN2 cDNA in the brain of children with LINCL. The vector consists of the AAV2 capsid enclosing the 4278-base single-stranded genome consisting of the two inverted terminal repeats of AAV serotype 2 and an expression cassette composed of the human cytomegalovirus (CMV) enhancer, the chicken beta-actin promoter/splice donor and 5' end of the intron, the 3' end of the rabbit P-globin intron and splice acceptor, the human CLN2 cDNA with an optimized Kozak translation initiation signal, and the polyadenylation/transcription stop codon from rabbit 3-globin. The proposed study will include 10 individuals and will be divided into two parts. Group A, to be studied first, will include four individuals with the severe form of the disease. Group B of the trial will include six individuals with a moderate form of the disease. After direct intracranial administration of the vector, there will be neurological assessment based on the LINCL clinical rating scale and magnetic resonance imaging/magnetic resonance spectroscopy assessment of the brain in regions of vector administration. The data generated will help evaluate two hypotheses: (1) that it is safe to carry out direct intracranial administration of the AAV2cuhCLN2 vector to the CNS of individuals with LINCL, and (2) that administration of the AAV2cuhCLN2 vector will slow down or halt the progression of the disease in the central nervous system.
Human Gene Therapy 12/2004; 15(11):1131-54. · 4.22 Impact Factor
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ABSTRACT: The mainstay of treatment for Parkinson's disease remains medical therapy. With improved surgical precision and decreased morbidity, stereotactic lesioning and deep brain stimulation have become more popular. New therapies currently in clinical trials include gene therapy and direct drug delivery to the brain. The present review discusses surgical therapies for the treatment of Parkinson's disease and the status of experimental strategies currently in preclinical and clinical testing.
Both stereotactic ablation and deep brain stimulation of the thalamus, globus pallidus interna, and subthalamic nucleus are discussed and compared in the current literature. New therapies such as drug infusions into the brain, gene therapy, and neural transplantation are in clinical trials and have been tested extensively in animals. Safety and efficacy of such therapies are discussed in recent literature.
Although medication remains the first and main line of treatment and the mainstay for Parkinson's disease, advances in techniques and safety of operations have made surgical interventions more popular. Thalamic surgery remains helpful only in a limited subset of patients with predominent tremor that is unresponsive to medication. Bilateral subthalamic nucleus DBS holds the most promising results for patients with tremor, severe motor fluctuations and dyskinesias from L-dopa, with the best improvements seen in daily activities and quality of life. Newer therapies currently in clinical trial include gene therapy to replace lost gamma-aminobutyric acid inputs to the subthalamic nucleus and globus pallidus interna/substantia nigra pars reticulata, and infusion of recombinant glial derived neurotrophic factor to support at-risk nigrostriatal neurons. Further developments in these areas, along with evolution in stem cell science that hopefully will permit replacement of lost neurons, may alter the nature of surgical practice in Parkinson's disease patients in the not too distant future.
Current Opinion in Neurology 09/2003; 16(4):487-93. · 4.94 Impact Factor
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ABSTRACT: A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB'), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential cis-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.
Journal of Virology 01/2003; 76(24):12792-802. · 5.40 Impact Factor
