The problems of delivering neuroactive molecules to the CNS

At present, the aetiologies of many neurological and neurodegenerative diseases are unknown. However, emergence of a better understanding of these diseases, at both cellular and molecular levels, opens up the possibility of replacement therapies. The presence of the blood-brain barrier complicates the delivery of molecules to the central nervous system. Numerous attempts have been made to bypass this barrier either by delivering the drugs directly into the brain or by transplanting cells to produce the missing molecules in situ. This review explores several methods for delivering bioactive molecules into the CNS, including the use of permeabilizers, osmotic pumps, slow polymer release systems and transplantation of cells with or without the use of the encapsulation technology.

A novel encapsulation device for peripheral implantation of antibody-secreting cells

Aurélien Lathuilière

Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly. As populations are aging in most parts of the world, the number of people with Alzheimer’s disease is expected to dramatically increase. This devastating neurodegenerative disorder remains the only major source of mortality without any effective disease-modifying treatment. Nevertheless, the progress made in understanding underlying disease mechanisms linked to amyloid and tau pathologies, led to the development of novel therapeutic strategies. The most clinically advanced experimental treatment is based on monoclonal antibodies (passive immunization) directed against the amyloid β peptide, a critical contributor to the neuropathology of Alzheimer’s disease. Although immunotherapy is able to clear cerebral amyloid deposits in patients, it has so far failed to slow down the loss of cognitive functions. In order to potentiate therapeutic effects, it may be critical to initiate the treatment before the first cognitive symptoms, and therefore chronically administer antibodies over years, possibly decades. This prompts the development of alternative strategies for the long-term delivery of therapeutic antibodies. To address this question, we have investigated an innovative approach based on a biologically active subcutaneous device encapsulating cells genetically engineered for the production of recombinant antibodies. In order to demonstrate that encapsulated cell technology can achieve peripheral continuous delivery of antibodies and lead to significant antibody exposure inside the brain, we rationally optimized parameters that are crucial to the rate of antibody secretion from such a cellular implant. In the first part of the thesis, we explored the utilization of lentiviral vectors for the genetic engineering of antibody secreting cell lines. We developed a dual vector strategy to significantly improve the efficiency of stable cell line generation. Moreover, the lentiviral transduction of C2C12 murine myoblasts allowed for the isolation of individual clones demonstrating high cell specific productivity, thus validating the use of C2C12 as a preclinical surrogate cell line for allogeneic implantation in disease mouse models. In the second part of the present work, we engineered a novel cell encapsulation device specially designed for subcutaneous implantation.We implemented a highly reproducible and scalable process to assemble devices supporting the implantation of large quantities of cells. Moreover, the in vivo optimization of initial cell seeding parameters enabled encapsulated cells to survive for months at high density inside the device. The final part of the present work assessed therapeutic efficacy of the newly developed cell encapsulation system for peripheral immunization against brain amyloid pathology. Devices secreting anti-Aβ antibodies were subcutaneously implanted in two transgenic mouse models of Alzheimer’s disease. Antibody levels in the mouse plasma reached 40 μg/ ml for several months, demonstrating long-term implant efficacy. Furthermore, we could demonstrate immunodecoration of amyloid plaques inside the brain with the delivered antibody, demonstrating penetration into the central nervous system. Remarkably, in the two mouse models of Alzheimer’s disease, brain amyloidosis was significantly reduced in the treated animals. Overall, this thesis work shows that peripheral implantation of encapsulated antibody-secreting myoblast cells is an effective strategy for passive immunization, particularly in the challenging context of delivering therapeutic amounts of antibodies to the central nervous system. Indeed, we demonstrate that anti-Aβ antibodies administered by subcutaneous bioactive implants can significantly decrease amyloid burden in mouse models of Alzheimer’s neuropathology.

EPFL2013

Adeno-Associated Virus-Mediated Gene Delivery for the Treatment of Amyotrophic Lateral Sclerosis and other Motor Neuron Disorders

Christopher Towne

The delivery of molecules and genes to the central nervous system (CNS) poses a major challenge for the treatment of neurodegenerative diseases. CNS disorders require long-term intervention and the presence of the blood-brain barrier (BBB) restricts the penetration of conventional drugs to the desired target cells. One approach towards stable delivery of therapeutic agents to the CNS is based on the transfer of DNA to target cells using viral vectors; a strategy known as gene therapy. Although viral vectors have provided encouraging results in CNS gene therapy trials for disorders such as Parkinson's disease, the small diffusion of the vector following direct injection into the brain region is not amenable to motor neuron (MN) disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy, that have cells distributed across fifty centimeters of spinal cord. The aim of this thesis was to explore non-invasive approaches for gene delivery to MNs of the spinal cord using adeno-associated viruses (AAV). AAV vectors are powerful gene transfer vehicles due to their low pathogenicity and ability to express genes in neurons for long periods of time. The first part of the thesis focused on ALS, an incurable paralytic disease resulting from a global death of MNs. Using a mouse model of ALS expressing a mutant form of superoxide dismutase 1 (SOD1) that causes the disease in humans, we have delivered genetic silencing instructions that act to degrade the SOD1 messenger RNA prior to its translation into the toxic protein. AAV serotype 6 was used to deliver the SOD1-silencing cassette through noninvasive peripheral routes to target relevant cell types in disease. The vector was administered intravenously following the hypothesis that AAV could infect MNs from the vasculature through traversing the peripheral axons that connect the MNs to the muscle fibers, a process known as retrograde transport. This resulted in infection of MNs across the spinal cord and brain stem, as well as almost total infection of skeletal muscle, another cell type implicated in ALS. Direct injections into multiple muscle groups were also performed, capitalizing on the retrograde transport capability of AAVs, to result in even higher levels of MN infection. These injections demonstrated that AAV serotype 6 could successfully deliver genes to MNs across the breadth of the spinal cord and resulted in protection of individual MN pools from ALS-mediated death. Curiously, however, despite the impressive infection profiles and neuroprotection at various spinal cord levels, neither of the techniques altered the disease course of the animals. These results stress the complexity of gene delivery and suggest that critical thresholds of SOD1-silencing and transduction across various cell types are required to rescue this particular disease model. The second component of the thesis concentrated purely on gene delivery to spinal cord neurons. AAV serotype 6 was injected directly into the muscles of Green African monkeys to determine whether the vector could undergo retrograde transport in larger animals. Indeed, efficient infection of MNs occurred, suggesting that this peripheral non-invasive delivery route is applicable to primates. These findings are not only relevant for human ALS therapy, but considering the similar dimensions of the injected monkeys with human newborns, directly relevant for treatment of infantile disease, spinal muscular atrophy. The last part of the thesis examined the ability of AAV serotype 6 to deliver genes to sensory neurons of spinal cord dorsal root ganglia in a mouse model of chronic pain. This study was initiated following the fortuitous observation of sensory neuron infection in the previous ALS mouse experiments. We examined various administration routes and found that AAV serotype 6 could efficiently deliver genes to sensory neurons in the context of chronic pain, demonstrating that the vector can be used to dissect mechanisms behind this disorder in rodents, and potentially serve as a vehicle for gene therapy towards intractable pain. In conclusion, the present thesis demonstrates that AAV serotype 6 is a powerful gene transfer vehicle for the CNS. Combined with future advances in AAV technology and delivery methods, the work presented here may one day facilitate gene therapy across the spinal cord for the treatment of devastating MN diseases, such as ALS and spinal muscular atrophy, as well as sensory neuron disorders, such as chronic pain.

EPFL2010

Transgenic SOD1 mice as a model for developing amyotrophic lateral sclerosis therapies

Sandrine Guillot

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a loss of motor neurons in the brain (brainstem and cortex) and the spinal cord that leads to a motor neurological symptomatology. Approximately 10% of ALS cases have a familial form of ALS. Among the familial cases, 20% are caused by dominantly inherited mutations (around 100 different mutations) in the protein Cu/Zn superoxide dismutase (SOD1). To date, genetic ALS models on mutated SOD1 (SOD1G93A, SOD1G85R and SOD1G37R) transgenesis in rodents have been successful in recapitulating the main features of the human pathology, especially the loss of spinal or facial motoneurons, the increased astrogliosis, the activation of microglia and the cellular and molecular disruptions in motoneurons. The therapies toward a treatment or cure for ALS have been met with limited success. In the present study, SOD1G93A transgenic mice have been used to test new gene therapies to slow disease progression and pharmacological approach to obtain a better comprehension of the pathological process, more precisely, to define the involvement of microglial activation in the spread of the disease. For the approach by gene therapy, injection of viral vectors in a localized region of central nervous system (CNS) constitutes an excellent tool for the development of new therapies. In the present study, the viral-mediated expression of potential neuroprotective factors was explored in the lumbar spinal cord or facial nucleus of SOD1G93A transgenic mice. As HIV-1-derived lentiviral vectors can efficiently transduce neurons in CNS, these retroviral vectors were used to over-express the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor) to supply a trophic support to lumbar and facial motoneurons, or RNAi molecules specifically targeting the SOD1G93A gene to inhibit the mutant SOD1 expression, the cause of the disease, in the lumbar spinal cord of SOD1G93A transgenic mice. For the study of the level of microglial involvement in the pathology, SOD1G93A transgenic mice had a daily pharmacological treatment with an inhibitor of microglial activation, the minocycline. The present thesis demonstrates that, on the one hand, at the difference of laboratories that delayed the disease progression by intramuscular injection of adenoviral vectors or adenoassociated vectors encoding for GDNF, the intraspinal injection of lentivirus encoding for GDNF did not lead to a protection of lumbar motoneurons and a delay of the disease. But this study showed a significantly rescue of facial motoneurons by the injection of lentivirus encoding for GDNF directly in facial nucleus. A selective vulnerability between motoneurons in facial nucleus and these in lumbar spinal cord has been therefore underlined. On the other hand, we showed that the therapeutic approach to inhibit the mutant SOD1 expression look as the most effective to delay the progression of the ALS pathology. Both studies of gene therapy, compared to others studies using the same therapeutic factors, demonstrated clearly that to optimize a therapeutic treatment, it seems better to act at muscular junction level with a retrograde transport form muscle to motoneuronal bodies of the therapeutic molecule or viral vector. Nevertheless, motoneuron-restricted silencing of mutant SOD1 would be useful for better understanding of neuronal and glial cellular mechanism mediating ALS-linked mutated SOD1 toxicity. Regarding the involvement of microglia in the pathological process, the inhibition of microglial activation by the minocycline treatment, suggests, on the one hand, that microglial activation do not initiate the pathological process and that this microgliosis might be a consequence and triggered by multiple disruptions. On the other hand, that minocycline could protect completely facial motoneurons and partially spinal motoneurons by its anti-apoptotic property. Therapeutic approaches aiming to act directly on the cause of the disease as well as a better comprehension of the role of non-neuronal cells in the pathological process may therefore open new perspectives for the treatment of ALS pathology.

EPFL2005