Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.

Develop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration.

Transthyretin (TTR) is a transport protein of retinol and thyroxine in serum and CSF, which is mainly secreted by liver and choroid plexus, and in smaller amounts in other cells throughout the body. The exact role of TTR and its specific expression in Central Nervous System (CNS) remains understudied. We investigated TTR expression and metabolism in CNS, through the intranasal and intracerebroventricular delivery of a specific anti-TTR Nanobody to the brain, unveiling Nanobody pharmacokinetics to the CNS. In TTR deficient mice, we observed that anti-TTR Nanobody was successfully distributed throughout all brain areas, and also reaching the spinal cord. In wild-type mice, a similar distribution pattern was observed. However, in areas known to be rich in TTR, reduced levels of Nanobody were found, suggesting potential target-mediated effects. Indeed, in wild-type mice, the anti-TTR Nanobody was specifically internalized in a receptor-mediated process, by neuronal-like cells, which were identified as motor neurons. Whereas in KO TTR mice Nanobody was internalized by all cells, for late lysosomal degradation. Moreover, we demonstrate that in vivo motor neurons also actively synthesize TTR. Finally, in vitro cultured primary motor neurons were also found to synthesize and secrete TTR into culture media. Thus, through a novel intranasal CNS distribution study with an anti-TTR Nanobody, we disclose a new cell type capable of synthesizing TTR, which might be important for the understanding of the physiological role of TTR, as well as in pathological conditions where TTR levels are altered in CSF, such as amyotrophic lateral sclerosis.

ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.

Transthyretin V30M mutation is the most common variant leading to Familial Amyloidotic Polyneuropathy. In this genetic disorder, Transthyretin accumulates preferentially in the extracellular matrix of peripheral and autonomic nervous systems leading to cell death and dysfunction. Thus, knowledge regarding important biological systems for Transthyretin clearance might unravel novel insights into Familial Amyloidotic Polyneuropathy pathophysiology. Herein, our aim was to evaluate the ability of glial cells from peripheral and autonomic nervous systems in Transthyretin uptake and degradation. We assessed the role of glial cells in Familial Amyloidotic Polyneuropathy pathogenesis with real-time polymerase chain reaction, immunohistochemistry, interference RNA and confocal microscopy.

Donnai-Barrow syndrome, a genetic disorder associated to LRP2 (low-density lipoprotein receptor 2/megalin) mutations, is characterized by unexplained neurological symptoms and intellectual deficits. Megalin is a multifunctional endocytic clearance cell-surface receptor, mostly described in epithelial cells. This receptor is also expressed in the CNS, mainly in neurons, being involved in neurite outgrowth and neuroprotective mechanisms. Yet, the mechanisms involved in the regulation of megalin in the CNS are poorly understood. Using transthyretin knockout mice, a megalin ligand, we found that transthyretin positively regulates neuronal megalin levels in different CNS areas, particularly in the hippocampus. Transthyretin is even able to rescue megalin downregulation in transthyretin knockout hippocampal neuronal cultures, in a positive feedback mechanism via megalin. Importantly, transthyretin activates a regulated intracellular proteolysis mechanism of neuronal megalin, producing an intracellular domain, which is translocated to the nucleus, unveiling megalin C-terminal as a potential transcription factor, able to regulate gene expression. We unveil that neuronal megalin reduction affects physiological neuronal activity, leading to decreased neurite number, length and branching, and increasing neuronal susceptibility to a toxic insult. Finally, we unravel a new unexpected role of megalin in synaptic plasticity, by promoting the formation and maturation of dendritic spines, and contributing for the establishment of active synapses, both in in vitro and in vivo hippocampal neurons. Moreover, these structural and synaptic roles of megalin impact on learning and memory mechanisms, since megalin heterozygous mice show hippocampal-related memory and learning deficits in several behaviour tests. Altogether, we unveil a complete novel role of megalin in the physiological neuronal activity, mainly in synaptic plasticity with impact in learning and memory. Importantly, we contribute to disclose the molecular mechanisms underlying the cognitive and intellectual disabilities related to megalin gene pathologies.

Inflammation is a hallmark of several neurodegenerative disorders including hereditary amyloidogenic transthyretin amyloidosis (ATTRv). ATTRv is an autosomal dominant neurodegenerative disorder with extracellular deposition of mutant transthyretin (TTR) aggregates and fibrils, particularly in nerves and ganglia of the peripheral nervous system. Nerve biopsies from ATTRv patients show increased cytokine production, but interestingly no immune inflammatory cellular infiltrate is observed around TTR aggregates. Here we show that as compared to Wild Type (WT) animals, the expression of several chemokines is highly downregulated in the peripheral nervous system of a mouse model of the disease. Interestingly, we found that stimulation of mouse Schwann cells (SCs) with WT TTR results in the secretion of several chemokines, a process that is mediated by toll-like receptor 4 (TLR4). In contrast, the secretion of all tested chemokines is compromised upon stimulation of SCs with mutant TTR (V30M), suggesting that V30M TTR fails to activate TLR4 signaling. Altogether, our data shed light into a previously unappreciated mechanism linking TTR activation of SCs and possibly underlying the lack of inflammatory response observed in the peripheral nervous system of ATTRv patients.