AAV based gene therapy for rare diseases

Of the seven thousand diseases that are described as rare, 80% of them have an identified genetic cause. With this in the mind, the development of technologies, such as gene therapy, to address the genetic factors involved in these pathologies, might be a game-changing therapeutic approach. Viral delivery of transgenes for RNA interference, DNA editing, or protein overexpression may offer opportunities for novel treatments. In this thesis, we developed adeno-associated virus (AAV) vector systems to tackle two rare diseases: Amyotrophic lateral sclerosis (ALS) caused by gain-of-function mutations in the superoxide dismutase 1 (SOD1) gene and inherited hearing loss disorder caused by mutation in the transmembrane channel-like 1 (TMC1) gene. ALS is a fatal neurodegenerative disease caused by the progressive degeneration of motoneurons (MN). Glial cells have been shown to importantly contribute to the disease. Silencing of the human pathogenic mutated SOD1 protein (SOD1G93A) in MN and/or astrocytes by AAV-mediated expression of microRNA targeting SOD1 has been shown to provide therapeutic benefits in a mouse model which is overexpressing SOD1G93A. However, in order to have an effective treatment, it is important to understand the influence of the therapy on several relevant ALS cell types. In this work, we determined the effects of lowering SOD1G93A expression in astrocytes using an AAV serotype 9, in combination with an astrocyte-specific promoter, in order to express a microRNA targeting SOD1. In the treated mice, we observed a significant improvement of the neuromuscular function and a partial protection of the vulnerable fast-fatigable MN. SOD1 silencing also induced a significant re-innervation of the neuromuscular junctions (NMJ) in the gastrocnemius muscle, observed after the initial phase of denervation, occurring around the age of 60 days. Re-innervation was associated with a grouping of the muscle fibers from the same type, consistent with enhanced axonal plasticity in the treated SOD1G93A mice. Overall, we demonstrated that silencing of SOD1 in astrocytes has an impact on MN plasticity at the level of NMJ, which translates into improved neuromuscular function towards the end stage of the disease. Mutations in the TMC1 gene lead to either complete deafness at birth, or to progressive hearing loss beginning in childhood, depending on the mode of inheritance (autosomal recessive or dominant, respectively). TMC1 is likely a component of the mechanotransduction channel, which is responsible for the transformation of mechanical sound-induced stimuli into electrical signals. This protein has been found to be expressed in the stereociliae of hair cells. In the second part of the thesis, we report on the development of viral tools to deliver a functional copy of the TMC1 gene, or its related paralog TMC2, into hair cells. We engineered several AAV vectors, in combination with different promoters, to identify a suitable combination able to restore TMC expression in the mouse cochlear hair cells, both in vitro and in vivo. We demonstrated, in collaboration with the lab of J. Holt, that when injected in TMC-null mice at birth, these vectors could partially restore loss of auditory function in this mouse model otherwise completely deaf. Overall we showed that AAV-based gene therapy is a promising approach to treat rare genetic disease.

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

Lentiviral and Adeno-Associated Vector-Based Therapy for Motor Neuron Disease Through RNAi

Patrick Aebischer, Christopher Towne

RNAi holds promise for neurodegenerative disorders caused by gain-of-function mutations. We and others have demonstrated proof-of-principle for viral-mediated RNAi in a mouse model of motor neuron disease. Lentivirus and adeno-associated virus have been used to knockdown levels of mutated superoxide dismutase 1 (SOD1) in the G93A SOD1 mouse model of familial amyotrophic lateral sclerosis (fALS) to result in beneficial therapeutic outcomes. This chapter describes the design, production, and titration of lentivirus and adeno-associated virus capable of mediating SOD1 knockdown in vivo. The delivery of the virus to the spinal cord directly, through intraspinal injection, or indirectly, through intramuscular injection, is also described, as well as the methods pertaining to the analysis of spinal cord transduction, SOD1 silencing, and determination of motor neuron protection

2009

Engineering of chitosan-based derivatives for non-viral gene delivery

Laura Camille Louise Nicolle

Gene therapy offers the possibility to treat or even cure diseases originating from genetic defects by introducing a therapeutic gene in target cells or correcting the initial defective gene. Amongst different options, non-viral vectors rely on the delivery of such genes using vehicles composed of lipids, inorganic nanoparticles or polymers. These systems are designed to protect the genetic material, while efficiently delivering it to the cells of interest. The initial backbone can be complemented with various components to help it overcome the many physiological barriers it may encounter during its journey. Amongst polymers, chitosan is a polysaccharide presenting many advantages for such purpose. Besides its biocompatibility and biodegradability, its potential for functionalization, thanks to numerous primary amines and hydroxyl groups, make it a backbone of choice for the preparation of a polymeric delivery vehicle. This work describes the covalent functionalization of chitosan with other polymers, peptides, and small molecules to later form polymeric nanoparticles condensing therapeutic DNA. Each component brings additional features to the initial system, thus finely tuning it for biological applications. The final system aims at treating liver inherited diseases such as ornithine transcarbamylase deficiency and phenylketonuria.This manuscript first exposes the preparation of a chitosan-based cationic system able to properly condense anionic DNA through electrostatic interactions. Due to its excellent DNA condensation properties, polyethylenimine was grafted to chitosan via a succinyl linker. The first in vitro investigations confirmed the potential of the resulting core system, but in vivo experiments highlighted toxicity coming from the remaining cationic charge on the nanoparticles. In order to better shield this cationic charge, bifunctional polyethylene glycol chains terminated by carboxylic acid moieties were grafted to chitosan amines. The resulting polymer was used to coat the first polymeric system through electrostatic interactions between the carboxylate groups and the remaining free amines of the chitosan-polyethylenimine derivative. The newly formed core-shell complex showed sustained cell viability in vitro and induced higher transfection efficiency of the gene as compared to the core system alone. However, its in vivo evaluation via retrograde intrabiliary infusion did not confirm such results: although transfection efficiency was retained, the core-shell complex did not significantly decrease the toxicity previously reported.The last part of this work includes the decoration of the core-shell system with targeting ligands making possible to perform in vivo assays via intravenous injection, for which the developed polymeric systems did not induce toxicity in preliminary experiments. So far, only a proof-of-concept with folic acid, an anti-cancer targeting ligand, was achieved. To do so, the shell polymer was further functionalized with folic acid-derived polyethylene glycol chains through strain-promoted azide-alkyne cycloaddition. The same strategy shall be applied in the near future with a liver-specific targeting ligand. Lastly, the improvement of the system's cell-penetration features was tackled by the grafting asparagusic acid-derived polyethylene glycol chains following the same strategy than for folic acid.