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

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.

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

Application of anti-amyloid beta immunization using encapsulated cell technology in mouse models of Alzheimer's disease

Vanessa Laversenne

Alzheimer's disease (AD) is the most common form of dementia in the elderly. AD is characterized by the deposition of two aggregated proteins: Amyloid beta (Aß) and hyperphosphorylated tau. Accumulations of these proteins are thought to be the signature of a pathogenic process causing the loss of neurons and synaptic connections associated with severe cognitive impairments in AD patients. Other important factors are involved in the neurodegeneration occurring in AD, such as neuroinflammation. In the present thesis work, we are investigating the effects of passive anti-Aß immunization. Passive anti-Aß immunization consists in the delivery of antibodies targeting Aß, with the aim to prevent its accumulation and promote the removal of pathological amyloid species. Although the treatment has been shown to clear amyloid pathology in the brain of AD patients, it has so far failed to prevent or slow down cognitive decline. Hence, it is important to determine the effects of anti-Aß immunization when administered either before or during Aß deposition, and explore how the treatment affects the Alzheimerâs manifestations in mouse models combining Aß and tau pathologies. In order to continuously deliver antibodies in AD mouse models, we used encapsulated cell technology (ECT) to implant myoblasts genetically engineered and secrete the recombinant mAb11 mouse IgG2a antibody directed against aggregated forms of Aß. Using this approach, we investigated the following three topics of research:

We examined the effect of a preventive administration of anti-Aß antibodies in a slowly progressing mouse model with both Aß and tau (P301L) pathologies. We demonstrated that delivery of anti-Aß antibodies before the onset of the disease dramatically reduces Aß pathology. We also found that the treatment decreases hyperphosphorylated tau accumulation and recruits microglial around Aß plaques.
We next assessed the effects of passive anti-Aß immunization in another AD mouse model overexpressing both Aß and wild-type (WT) tau. In this second study, we administered the anti-Aß antibodies when both Aß and tau pathologies were already established. In the hippocampal formation, we showed that WT tau overexpression might affect the microglial response to ongoing pathology by reducing the tight interaction of these cells with the amyloid plaques. This effect, however, is counteracted by passive anti-Aß immunization, which was found to redirect microglia towards Aß deposits. In addition, passive anti-Aß immunization decreases tau spreading throughout the hippocampus, but fails to prevent neither tau phosphorylation, nor the hippocampal degeneration induced by local tau overexpression.
In parallel, we developed a human myoblast cell line secreting anti-Aß antibodies for translation of our ECT towards human application. Using C2C12 mouse myoblasts, our flat-sheet device allowed for long-term continuous antibody delivery in the plasma of subcutaneously implanted mice. We were able to generate antibody-secreting human cells using the immortalized C25 cell It was possible to differentiate these cells into myotubes and adapt them to long-term survival inside the capsules maintained in vitro. However, when implanted in the subcutaneous tissue of immunocompromised mice, myoblasts survived in the ECT device as a thin layer of cells and were not able to secrete recombinant antibodies in sufficient amounts to be considered for therapeutic purpose.

EPFL2019