Marike Broekman

Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain.

Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8 x 10(10) genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.

AAV-Mediated Gene Delivery in Adult GM1- Gangliosidosis Mice Corrects Lysosomal Storage in CNS and Improves Survival

Background GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid β-galactosidase (βgal), which results in the accumulation of GM1-ganglioside and its asialo-form (GA1) primarily in the CNS. Age of onset ranges from infancy to adulthood, and excessive ganglioside accumulation produces progressive neurodegeneration and psychomotor retardation in humans. Currently, there are no effective therapies for the treatment of GM1-gangliosidosis. Methodology/Principal Findings In this study we examined the effect of thalamic infusion of AAV2/1-βgal vector in adult GM1 mice on enzyme distribution, activity, and GSL content in the CNS, motor behavior, and survival. Six to eight week-old GM1 mice received bilateral injections of AAV vector in the thalamus, or thalamus and deep cerebellar nuclei (DCN) with pre-determined endpoints at 1 and 4 months post-injection, and the humane endpoint, or 52 weeks of age. Enzyme activity was elevated throughout the CNS of AAV-treated GM1 mice and GSL storage nearly normalized in most structures analyzed, except in the spinal cord which showed ∼50% reduction compared to age-matched untreated GM1 mice spinal cord. Survival was significantly longer in AAV-treated GM1 mice (52 wks) than in untreated mice. However the motor performance of AAV-treated GM1 mice declined over time at a rate similar to that observed in untreated GM1 mice. Conclusions/Significance Our studies show that the AAV-modified thalamus can be used as a ‘built-in’ central node network for widespread distribution of lysosomal enzymes in the mouse cerebrum. In addition, this study indicates that thalamic delivery of AAV vectors should be combined with additional targets to supply the cerebellum and spinal cord with therapeutic levels of enzyme necessary to achieve complete correction of the neurological phenotype in GM1 mice.

Valproic acid for the treatment of malignant gliomas: review of the preclinical rationale and published clinical results.

Introduction: Glioblastoma multiforme is the most common and aggressive primary brain tumor. Valproate has been used as an anti-epileptic drug and mood stabilizer for decades. Recently, it was found to inhibit the proliferation of various cancers including glioblastoma multiforme. Areas covered: We provide a comprehensive review of the mechanisms of action of valproate in gliomas, of its potential side effects and of the published clinical results obtained with this drug in glioblastomas. Valproate inhibits a subset of histone deacetylases and cellular kinases, and affects gene transcription through histone hyperacetylation, DNA hypomethylation and the modulation of several transcription factors. As a result, VPA induces differentiation of glioma cells, can prevent their invasion in surrounding tissues and may inhibit tumor angiogenesis. VPA can also inhibit DNA repair, thereby potentiating cytotoxic treatments such as chemotherapies or radiation therapy. Based on these mechanisms and case reports of glioblastoma remissions following VPA treatment, several clinical studies currently assess the therapeutic potential of VPA in glioma therapy. Expert opinion: The combination of VPA treatment with chemotherapy and radiotherapy in glioblastoma appears a rational option that deserves well-designed prospective clinical trials that assess the efficacy and the molecular characteristics of the responding tumors in these patients.

Preventing Growth of Brain Tumors by Creating a Zone of Resistance

Glioblastoma multiforme (GBM) is a devastating form of brain cancer for which there is no effective treatment. Here, we report a novel approach to brain tumor therapy through genetic modification of normal brain cells to block tumor growth and effect tumor regression. Previous studies have focused on the use of vector-based gene therapy for GBM by direct intratumoral injection with expression of therapeutic proteins by tumor cells themselves. However, as antitumor proteins are generally lethal to tumor cells, the therapeutic reservoir is rapidly depleted, allowing escape of residual tumor cells. Moreover, it has been difficult to achieve consistent transduction of these highly heterogeneous tumors. In our studies, we found that transduction of normal cells in the brain with an adeno-associated virus (AAV) vector encoding interferon-beta (IFN-beta) was sufficient to completely prevent tumor growth in orthotopic xenograft models of GBM, even in the contralateral hemisphere. In addition, complete eradication of established tumors was achieved through expression of IFN-beta by neurons using a neuronal-restricted promoter. To our knowledge this is the first direct demonstration of the efficacy of targeting gene delivery exclusively to normal brain cells for brain tumor therapy.

Quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including ‘microvesicles’ and ‘exosomes’, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging. This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Mechanisms of distribution of mouse |[beta]|-galactosidase in the adult GM1-gangliosidosis brain

GM1-gangliosidosis is a lysosomal storage disease (LSD) caused by an autosomal recessive deficiency of lysosomal acid β-galactosidase (βgal). This leads to accumulation of GM1-ganglioside and its asialo derivative GA1 in the central nervous system (CNS), and progressive neurodegeneration. Therapeutic AAV-mediated gene delivery to the brain for LSDs has proven very successful in several animal models. GM1-gangliosidosis is also a prime candidate for AAV-mediated gene therapy in the CNS. As global neuropathology characterizes the most severe forms of this disease, therapeutic interventions need to achieve distribution of βgal throughout the entire CNS. Therefore, careful consideration of routes of administration and target structures from where metabolically active enzyme can be produced, released and distributed throughout the CNS, is necessary. The goal of this study was to investigate the pattern and mechanism of distribution of βgal in the adult GM1-gangliosidosis mouse brain upon hippocampal injection of an AAV vector-encoding βgal. We found evidence that three different mechanisms contribute to its distribution in the brain: (1) diffusion; (2) axonal transport within neurons from the site of production; (3) CSF flow in the perivascular space of Virchow–Robin. In addition, we found evidence of axonal transport of vector-encoded mRNA.

Prognostic relevance of epilepsy at presentation in glioblastoma patients.

BACKGROUND Epileptogenic glioblastomas are thought to convey a favorable prognosis, either due to early diagnosis or potential antitumor effects of antiepileptic drugs. We investigated the relationship between survival and epilepsy at presentation, early diagnosis, and antiepileptic drug therapy in glioblastoma patients. METHODS Multivariable Cox regression was applied to survival data of 647 consecutive patients diagnosed with de novo glioblastoma between 2005 and 2013 in order to investigate the association between epilepsy and survival in glioblastoma patients. In addition, we quantified the association between survival and valproic acid (VPA) treatment. RESULTS Epilepsy correlated positively with survival (HR: 0.75 (95% CI: 0.61-0.92), P < .01). This effect is independent of age, sex, performance status, type of surgery, adjuvant therapy, tumor location, and tumor volume, suggesting that this positive correlation cannot be attributed solely to early diagnosis. For patients who presented with epilepsy, the use of the antiepileptic drug VPA did not associate with survival when compared with patients who did not receive VPA treatment. CONCLUSION Epilepsy is an independent prognostic factor for longer survival in glioblastoma patients. This prognostic effect is not solely explained by early diagnosis, and survival is not associated with VPA treatment.

Ethical clinical translation of stem cell interventions for neurologic disease

The application of stem cell transplants in clinical practice has increased in frequency in recent years. Many of the stem cell transplants in neurologic diseases, including stroke, Parkinson disease, spinal cord injury, and demyelinating diseases, are unproven—they have not been tested in prospective, controlled clinical trials and have not become accepted therapies. Stem cell transplant procedures currently being carried out have therapeutic aims, but are frequently experimental and unregulated, and could potentially put patients at risk. In some cases, patients undergoing such operations are not included in a clinical trial, and do not provide genuinely informed consent. For these reasons and others, some current stem cell interventions for neurologic diseases are ethically dubious and could jeopardize progress in the field. We provide discussion points for the evaluation of new stem cell interventions for neurologic disease, based primarily on the new Guidelines for Stem Cell Research and Clinical Translation released by the International Society for Stem Cell Research in May 2016. Important considerations in the ethical translation of stem cells to clinical practice include regulatory oversight, conflicts of interest, data sharing, the nature of investigation (e.g., within vs outside of a clinical trial), informed consent, risk-benefit ratios, the therapeutic misconception, and patient vulnerability. To help guide the translation of stem cells from the laboratory into the neurosurgical clinic in an ethically sound manner, we present an ethical discussion of these major issues at stake in the field of stem cell clinical research for neurologic disease.

The history of head transplantation: a review

BackgroundSince the turn of the last century, the prospect of head transplantation has captured the imagination of scientists and the general public. Recently, head transplant has regained attention in popular media, as neurosurgeons have proposed performing this procedure in 2017. Given the potential impact of such a procedure, we were interested in learning the history of the technical hurdles that need to be overcome, and determine if it is even technically possible to perform such a procedure on humans today.MethodWe conducted a historical review of available literature on the technical challenges and developments of head transplantation. The many social, psychological, ethical, religious, cultural, and legal questions of head transplantation were beyond the scope of this review.ResultsOur historical review identified the following important technical considerations related to performing a head transplant: maintenance of blood flow to an isolated brain via vessel anastomosis; availability of immunosuppressive agents; spinal anastomosis and fusion following cord transfection; pain control in the recipient. Several animal studies have demonstrated success in maintaining recipient cerebral perfusion and achieving immunosuppression. However, there is currently sparse evidence in favor of successful spinal anastomosis and fusion after transection. While recent publications by an Italian group offer novel approaches to this challenge, research on this topic has been sparse and hinges on procedures performed in animal models in the 1970s. How transferrable these older methods are to the human nervous system is unclear and warrants further exploration.ConclusionsOur review identified several important considerations related to performing a viable head transplantation. Besides the technical challenges that remain, there are important ethical issues to consider, such as exploitation of vulnerable patients and informed consent. Thus, besides the remaining technical challenges, these ethical issues will also need to be addressed before moving these studies to the clinic.

Surgical innovation : The ethical agenda

AbstractThe aim of the present article was to systematically review the ethics of surgical innovation and introduce the components of the learning health care system to guide future research and debate on surgical innovation.Although the call for evidence-based practice in surgery is increasingly high on the agenda, most surgeons feel that the format of the randomized controlled trial is not suitable for surgery. Innovation in surgery has aspects of, but should be distinguished from both research and clinical care and raises its own ethical challenges.To answer the question “What are the main ethical aspects of surgical innovation?”, we systematically searched PubMed and Embase. Papers expressing an opinion, point of view, or position were included, that is, normative ethical papers.We included 59 studies discussing ethical aspects of surgical innovation. These studies discussed 4 major themes: oversight, informed consent, learning curve, and vulnerable patient groups. Although all papers addressed the ethical challenges raised by surgical innovation, surgeons hold no uniform view of surgical innovation, and there is no agreement on the distinction between innovation and research. Even though most agree to some sort of oversight, they offer different alternatives ranging from the formation of new surgical innovation committees to establishing national registries. Most agree that informed consent is necessary for innovative procedures and that surgeons should be adequately trained to assure their competence to tackle the learning curve problem. All papers agree that in case of vulnerable patients, alternatives must be found for the informed consent procedure.We suggest that the concept of the learning health care system might provide guidance for thinking about surgical innovation. The underlying rationale of the learning health care system is to improve the quality of health care by embedding research within clinical care. Two aspects of a learning health care system might particularly enrich the necessary future discussion on surgical innovation: integration of research and practice and a moral emphasis on “learning activities.” Future research should evaluate whether the learning health care system and its adjacent moral framework provides ethical guidance for evidence-based surgery.