Current state of Health Canada regulation for cellular and gene therapy products: potential cures on the horizon.

We provide an overview of the regulatory framework, pathways and underlying regulatory authority for cell, gene and tissue-engineered therapies in Canada. Canada's regulatory approach uses three sets of regulations, namely, the Cells, Tissues and Organs Regulations, the Food and Drug Regulations and the Medical Devices Regulations. We provide an overview of each these sets of regulations as they apply to clinical investigation to post-market product lifecycle stages. Information is provided on the current sources of relevant Health Canada guidance documents. We highlight several regional success stories including Prochymal, a cell therapy product that achieved Canadian regulatory approval using the conditional marketing approval system. We also examine the perceived gaps in the Canadian regulations and how those gaps are being addressed by interactions between the government, stakeholders and international bodies. We conclude that the risk-benefit approach used by Health Canada for regulatory approval processes is sufficiently flexible to enable to development of novel cell and gene therapy products in Canada, yet stringent enough to protect patient safety.

The extent of intrauterine growth restriction determines the severity of cerebral injury and neurobehavioural deficits in rodents.

BACKGROUND: Cerebral Palsy (CP) is the most common physical pediatric neurodevelopmental disorder and spastic diplegic injury is its most frequent subtype. CP results in substantial neuromotor and cognitive impairments that have significant socioeconomic impact. Despite this, its underlying pathophysiological mechanisms and etiology remain incompletely understood. Furthermore, there is a need for clinically relevant injury models, which a) reflect the heterogeneity of the condition and b) can be used to evaluate new translational therapies. To address these key knowledge gaps, we characterized a chronic placental insufficiency (PI) model, using bilateral uterine artery ligation (BUAL) of dams. This injury model results in intrauterine growth restriction (IUGR) in pups, and animals recapitulate the human phenotype both in terms of neurobehavioural and anatomical deficits. METHODS: Effects of BUAL were studied using luxol fast blue (LFB)/hematoxylin & eosin (H&E) staining, immunohistochemistry, quantitative Magnetic Resonance Imaging (MRI), and Catwalk neurobehavioural tests. RESULTS: Neuroanatomical analysis revealed regional ventricular enlargement and corpus callosum thinning in IUGR animals, which was correlated with the extent of growth restriction. Olig2 staining revealed reductions in oligodendrocyte density in white and grey matter structures, including the corpus callosum, optic chiasm, and nucleus accumbens. The caudate nucleus, along with other brain structures such as the optic chiasm, internal capsule, septofimbrial and lateral septal nuclei, exhibited reduced size in animals with IUGR. The size of the pretectal nucleus was reduced only in moderately injured animals. MAG/NF200 staining demonstrated reduced myelination and axonal counts in the corpus callosum of IUGR animals. NeuN staining revealed changes in neuronal density in the hippocampus and in the thickness of hippocampal CA2 and CA3 regions. Diffusion weighted imaging (DWI) revealed regional white and grey matter changes at 3 weeks of age. Furthermore, neurobehavioural testing demonstrated neuromotor impairments in animals with IUGR in paw intensities, swing speed, relative print positions, and phase dispersions. CONCLUSIONS: We have characterized a rodent model of IUGR and have demonstrated that the neuroanatomical and neurobehavioural deficits mirror the severity of the IUGR injury. This model has the potential to be applied to examine the pathobiology of and potential therapeutic strategies for IUGR-related brain injury. Thus, this work has potential translational relevance for the study of CP.

Community engagement and knowledge translation: progress and challenge in autism research.

The last decade has seen significant growth in scientific understanding and public awareness of autism. There is still a long road ahead before this awareness can be matched with parallel improvements in evidence-based practice. The process of translating evidence into community care has been hampered by the seeming disconnect between the mainstream scientific research agenda and the immediate priorities of many communities. The need for community engagement in the process of translating knowledge into impact has been recognized. However, there remains little consensus or empirical data regarding the process of such engagement and how to measure its impact. We shed light on a number of engagement models and tools, previously advocated in health research, as they apply to autism research. Furthermore, we illustrate the utility of such tools in supporting identification of knowledge gaps and priorities, using two community-based case studies. The case studies illustrate that information generated from research is indeed relevant and critical for knowledge users in the community. Simple and systematic methods can support the translation and uptake of knowledge in diverse communities, therefore enhancing engagement with research and bridging research findings with immediate community needs.

The potential for stem cells in cerebral palsy--piecing together the puzzle.

The substantial socioeconomic burden of a diagnosis of cerebral palsy, coupled with a positive anecdotal and media spin on stem cell treatments, drives many affected families to seek information and treatment outside of the current clinical and scientific realm. Preclinical studies using several types of stem and adult cells--including mesenchymal stem cells, neural precursor cells, olfactory ensheathing glia and Schwann cells--have demonstrated some regenerative and functional efficacy in neurologic paradigms. This paper describes the most common cell types investigated for transplant in vivo and summarizes the current state of early-phase clinical trials. It investigates the most relevant and promising coadministered therapies, including rehabilitation, drug targeting, magnetic stimulation, and bioengineering approaches. We highlight the need for adjunctive combinatorial strategies to successfully transfer stem cell treatments from bench to bedside.

Effects of adult neural precursor-derived myelination on axonal function in the perinatal congenitally dysmyelinated brain: optimizing time of intervention, developing accurate prediction models, and enhancing performance.

Stem cell repair shows substantial translational potential for neurological injury, but the mechanisms of action remain unclear. This study aimed to investigate whether transplanted stem cells could induce comprehensive functional remyelination. Subventricular zone (SVZ)-derived adult neural precursor cells (aNPCs) were injected bilaterally into major cerebral white matter tracts of myelin-deficient shiverer mice on postnatal day (P) 0, P7, and P21. Tripotential NPCs, when transplanted in vivo, integrated anatomically and functionally into local white matter and preferentially became Olig2+, Myelin Associated Glycoprotein-positive, Myelin Basic Protein-positive oligodendrocytes, rather than Glial Fibrillary Acidic Protein-positive astrocytes or Neurofiliment 200-positive neurons. Processes interacted with axons and transmission electron microscopy showed multilamellar axonal ensheathment. Nodal architecture was restored and by quantifying these anatomical parameters a computer model was generated that accurately predicted action potential velocity, determined by ex vivo slice recordings. Although there was no obvious phenotypic improvement in transplanted shi/shis, myelinated axons exhibited faster conduction, lower activation threshold, less refractoriness, and improved response to high-frequency stimulation than dysmyelinated counterparts. Furthermore, they showed improved resilience to ischemic insult, a promising finding in the context of perinatal brain injury. This study describes, for the first time mechanistically, the functional characteristics and anatomical integration of nonimmortalized donor SVZ-derived murine aNPCs in the dysmyelinated brain at key developmental time points.

The potential for stem cell therapies to have an impact on cerebral palsy: opportunities and limitations.

Cerebral palsy (CP) is a chronic childhood disorder described by a group of motor and cognitive impairments and results in a substantial socio-economic burden to the individual, family, and healthcare system. With no effective biological interventions, therapies for CP are currently restricted to supportive and management strategies. Cellular transplantation has been suggested as a putative intervention for neural pathology, as mesenchymal and neural stem cells, as well as olfactory ensheathing glia and Schwann cells, have shown some regenerative and functional efficacy in experimental central nervous system disorders. This review describes the most common cell types investigated and delineates their purported mechanisms in vivo. Furthermore, it provides a cogent summary of both current early-phase clinical trials using neural precursor cells (NPCs) and the state of stem cell therapies for neurodegenerative conditions. Although NPCs are perhaps the most promising candidates for cell replacement therapy in the context of CP, much still remains to be understood regarding safety, efficacy, timing, dose, and route of transplantation, as well as the capacity for combinatorial strategies.

Development and maturation of the spinal cord: implications of molecular and genetic defects.

The human central nervous system (CNS) may be the most complex structure in the universe. Its development and appropriate specification into phenotypically and spatially distinct neural subpopulations involves a precisely orchestrated response, with thousands of transcriptional regulators combining with epigenetic controls and specific temporal cues in perfect synchrony. Understandably, our insight into the sophisticated molecular mechanisms which underlie spinal cord development are as yet limited. Even less is known about abnormalities of this process - putative genetic and molecular causes of well-described defects have only begun to emerge in recent years. Nonetheless, modern scientific techniques are beginning to demonstrate common patterns and principles amid the tremendous complexity of spinal cord development and maldevelopment. These advances are important, given that developmental anomalies of the spinal cord are an important cause of mortality and morbidity (Sadler, 2000); it is hoped that research advances will lead to better methods to detect, treat, and prevent these lesions.

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate.

Although neural c-Jun is essential for successful peripheral nerve regeneration, the cellular basis of this effect and the impact of c-Jun activation are incompletely understood. In the current study, we explored the effects of neuron-selective c-Jun deletion, substitution of serine 63 and 73 phosphoacceptor sites with non-phosphorylatable alanine, and deletion of Jun N-terminal kinases 1, 2 and 3 in mouse facial nerve regeneration. Removal of the floxed c-jun gene in facial motoneurons using cre recombinase under control of a neuron-specific synapsin promoter (junΔS) abolished basal and injury-induced neuronal c-Jun immunoreactivity, as well as most of the molecular responses following facial axotomy. Absence of neuronal Jun reduced the speed of axonal regeneration following crush, and prevented most cut axons from reconnecting to their target, significantly reducing functional recovery. Despite blocking cell death, this was associated with a large number of shrunken neurons. Finally, junΔS mutants also had diminished astrocyte and microglial activation and T-cell influx, suggesting that these non-neuronal responses depend on the release of Jun-dependent signals from neighboring injured motoneurons. The effects of substituting serine 63 and 73 phosphoacceptor sites (junAA), or of global deletion of individual kinases responsible for N-terminal c-Jun phosphorylation were mild. junAA mutants showed decrease in neuronal cell size, a moderate reduction in post-axotomy CD44 levels and slightly increased astrogliosis. Deletion of Jun N-terminal kinase (JNK)1 or JNK3 showed delayed functional recovery; deletion of JNK3 also interfered with T-cell influx, and reduced CD44 levels. Deletion of JNK2 had no effect. Thus, neuronal c-Jun is needed in regeneration, but JNK phosphorylation of the N-terminus mostly appears to not be required for its function.

Cell-based transplantation strategies to promote plasticity following spinal cord injury.

Cell transplantation therapy holds potential for repair and functional plasticity following spinal cord injury (SCI). Stem and progenitor cells are capable of modifying the lesion environment, providing structural support and myelination and increasing neurotrophic factors for neuroprotection and endogenous activation. Through these effects, transplanted cells induce plasticity in the injured spinal cord by promoting axonal elongation and collateral sprouting, remyelination, synapse formation and reduced retrograde axonal degeneration. In light of these beneficial effects, cell transplantation could be combined with other treatment modalities, such as rehabilitation and immune modulation, to provide a synergistic functional benefit. This review will delineate 1) stem/progenitor cell types proposed for cell transplantation in SCI, 2) in vitro evidence of cell-induced mechanisms of plasticity, 3) promotion of functional recovery in animal models of SCI, 4) successful combinatorial strategies using cell transplantation. Current treatment modalities for SCI provide modest efficacy, especially in chronic stages of SCI. Hence, combinatorial stem cell transplantation strategies which could potentially directly address tissue sparing and neuroplasticity in chronic SCI show promise. Rigorous evaluation of combinatorial approaches using stem cell transplantation with appropriate preclinical animal models of SCI is needed to advance therapeutic strategies to the point where clinical trials are appropriate. Given the high patient demand for and clinical trial precedent of cell transplantation therapy, combination stem cell therapies have the promise to provide improved quality of life for individuals, with corresponding socioeconomic benefit.

Acute postischemic seizures are associated with increased mortality and brain damage in adult mice.

Postischemic seizures are associated with worsened outcome following stroke, but the underlying pathophysiology is poorly understood. Here we examined acute seizures in adult mice following hypoxia-ischemia (HI) via combined behavioral, electrophysiological, and histological assessments. C57BL/6 mice aged 4-9 months received a permanent occlusion of the right common carotid artery and then underwent a systemic hypoxic episode. Generalized motor seizures were observed within 72 h following HI. These seizures occurred nearly exclusively in animals with extensive brain injury in the hemisphere ipsilateral to the carotid occlusion, but their generation was not associated with electroencephalographic discharges in bilateral hippocampal and neocortical recordings. Animals exhibiting these seizures had a high rate of mortality, and post-HI treatments with diazepam and phenytoin greatly suppressed these behavioral seizures and improved post-HI animal survival. Based on these data, we conclude that these seizures are a consequence of HI brain injury, contribute to worsened outcome following HI, and that they originate from deep subcortical structures.

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: time course and molecular characterization.

Generation of new axonal sprouts plays an important role in neural repair. In the current study, we examined the appearance, composition and effects of gene deletions on intrabrainstem sprouts following peripheral facial nerve axotomy. Axotomy was followed by the appearance of galanin(+) and calcitonin gene-related peptide (CGRP)(+) sprouts peaking at day 14, matching both large, neuropeptide(+) subpopulations of axotomized facial motoneurons, but with CGRP(+) sprouts considerably rarer. Strong immunoreactivity for vesicular acetylcholine transporter (VAChT) and retrogradely transported MiniRuby following its application on freshly cut proximal facial nerve stump confirmed their axotomized motoneuron origin; the sprouts expressed CD44 and alpha7beta1 integrin adhesion molecules and grew apparently unhindered along neighboring central white matter tracts. Quantification of the galanin(+) sprouts revealed a stronger response following cut compared with crush (day 7-14) as well as enhanced sprouting after recut (day 8 + 6 vs. 14; 14 + 8 vs. 22), arguing against delayed appearance of sprouting being the result of the initial phase of reinnervation. Sprouting was strongly diminished in brain Jun-deficient mice but enhanced in alpha7 null animals that showed apparently compensatory up-regulation in beta1, suggesting important regulatory roles for transcription factors and the sprout-associated adhesion molecules. Analysis of inflammatory stimuli revealed a 50% reduction 12-48 hours following systemic endotoxin associated with neural inflammation and a tendency toward more sprouts in TNFR1/2 null mutants (P = 10%) with a reduced inflammatory response, indicating detrimental effects of excessive inflammation. Moreover, the study points to the usefulness of the facial axotomy model in exploring physiological and molecular stimuli regulating central sprouting.