Development of lentiviral vectors targeted to P38MAPK inhibition and Akt activation in motor neurons of a mouse model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by progressive loss of motor neurons in the cortex, brainstem and spinal cord. This leads to weakness and muscular atrophy that evolves to paralysis and death. Studies on patients and animal models of ALS suggest that the pathology is the consequence of the unbalance between pro-degenerative (p38MAPK) and pro-survival (Akt) pathways in motor neurons. In order to re-equilibrate this complex interplay, the major objective of this study was to develop a gene-based approach to target, in vivo, these two intracellular signaling proteins. We demonstrated for the first time that a construct derived from Hb9 promoter can be used in lentivectors to restrict transgene expression to motor neurons in vivo; moreover we found that induction of Akt pathway, through expression of constitutively activated Akt3 in motor neurons, prevents neuronal loss in SOD1G93A mice, although it does not influence their premature death. On the other hand, we used RNAi to downregulate p38MAPKalpha in a mouse model of ALS. We revealed that expression of p38MAPK-targeted shRNA in the spinal cord of presymptomatic mice reduces motor neuronal loss in the early phases of the pathology, but then it is not able to sustain neuronal survival during disease progression, eventually resulting in worsening of symptoms and shortening of life span. We found preliminary evidences that hyperactivation of microglial cells may be responsible for this more aggressive phenotype, suggesting that a fine tuning of microglial reactivity may be important to avoid pro-degenerative effects. Overall these data support the hypothesis that modulation of pro-degenerative and pro-survival pathways may help counteracting motor neuronal degeneration, and strengthen the evidence that preservation of neuronal perikaria is not sufficient to ameliorate disease progression in ALS. The peripheral compartment (axons, muscles) should be regarded as an additional target for potential therapeutic approaches

Synthesis of zwitterionic-functionalized conjugated nanoparticles for targeted drug delivery applications

Polymeric Nanoparticles (NPs) represent a promising pharmacological tool, since their structure can be modified to obtain: i) encapsulation and controlled release of a wide range of active compounds, ranging from small molecules to siRNA or oligonucleotides; ii) selective cell targeting, thus allowing precise drug delivery to the desired site of action. A powerful strategy to achieve selectivity of uptake in specific cell types is to conjugate the nanoparticles to a ligand specific for receptors expressed by the target cell type. This offers the advantage of a potentially improved drug efficacy with limited side effects and toxicity. Polymeric nanoparticles in a range of 20–100 nm have a high potential for in vivo applications, due to their ability to circulate in the blood for a long period of time. In fact, this size range allows to avoid renal and lymphatic clearance, to prevent opsonization and at the same time improves the internalization by cells. In this work we address the synthesis by reversible addition-fragmentation chain transfer (RAFT) of biodegradable, zwitterionic-based nanoparticles. This Zwitterionic nanoparticles act as super non-fouling surfaces that prevent protein adsorption from complex biological media. The nanoparticles were functionalized with different numbers of selective ligands through click chemistry; different dimensions were synthetized changing the length of the hydrophobic part. In vitro studies were performed to evaluate the uptake of functionalized nanoparticles

Development of lentiviral vectors targeted to p38MAPK inhibition and Akt activation in motor neurons of a mouse model of familial amyotrophic laterial sclerosis

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Delivery Platforms for CRISPR/Cas9 Genome Editing of Glial Cells in the Central Nervous System

: Glial cells (astrocytes, oligodendrocytes, and microglia) are emerging as key players in several physiological and pathological processes of the central nervous system (CNS). Astrocytes and oligodendrocytes are not only supportive cells that release trophic factors or regulate energy metabolism, but they also actively modulate critical neuronal processes and functions in the tripartite synapse. Microglia are defined as CNS-resident cells that provide immune surveillance; however, they also actively contribute to shaping the neuronal microenvironment by scavenging cell debris or regulating synaptogenesis and pruning. Given the many interconnected processes coordinated by glial cells, it is not surprising that both acute and chronic CNS insults not only cause neuronal damage but also trigger complex multifaceted responses, including neuroinflammation, which can critically contribute to the disease progression and worsening of symptoms in several neurodegenerative diseases. Overall, this makes glial cells excellent candidates for targeted therapies to treat CNS disorders. In recent years, the application of gene editing technologies has redefined therapeutic strategies to treat genetic and age-related neurological diseases. In this review, we discuss the advantages and limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based gene editing in the treatment of neurodegenerative disorders, focusing on the development of viral- and nanoparticle-based delivery methods for in vivo glial cell targeting