Modular Multifunctional Protein Vectors for Gene Therapy

Sin resumen disponibleAgencia Nacional de Investigación e Innovació

Una llum d'esperança : alumnes repetidors com a monitors de pràctiques. Experiència a segon curs de la Llicenciatura en Medicina

Amb l'ànim d'ajudar als estudiants que tenen problemes a la assignatura "Estructura Microscòpica d'Aparells i Sistemes Humans" de 2n curs de la llicenciatura de Medicina i que la suspenen reiteradamet, el curs 2005-2006 vam iniciar un nou projecte d'innovació docent basat en l'èxit de l'experiència dels professors i monitors de l'assignatura de Neuroanatomia de segon curs de Llicenciatura en medicina durant el curs 2004-2005

Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

Background: Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated. Results: Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced. Conclusion: When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene

BBB-targeting, protein-based nanomedicines for drug and nucleic acid delivery to the CNS

The increasing incidence of diseases affecting the central nervous system (CNS) demands the urgent development of efficient drugs. While many of these medicines are already available, the Blood Brain Barrier and to a lesser extent, the Blood Spinal Cord Barrier pose physical and biological limitations to their diffusion to reach target tissues. Therefore, efforts are needed not only to address drug development but specially to design suitable vehicles for delivery into the CNS through systemic administration. In the context of the functional and structural versatility of proteins, recent advances in their biological fabrication and a better comprehension of the physiology of the CNS offer a plethora of opportunities for the construction and tailoring of plain nanoconjugates and of more complex nanosized vehicles able to cross these barriers. We revise here how the engineering of functional proteins offers drug delivery tools for specific CNS diseases and more transversally, how proteins can be engineered into smart nanoparticles or 'artificial viruses' to afford therapeutic requirements through alternative administration routes

The Receptor CMRF35-Like Molecule-1 (CLM-1) Enhances the Production of LPS-Induced Pro-Inflammatory Mediators during Microglial Activation.

CMRF35-like molecule-1 (CLM-1) belongs to a receptor family mainly expressed in myeloid cells that include activating and inhibitory receptors. CLM-1 contains two ITIMs and a single immunoreceptor tyrosine-based switch motif (ITSM), although also displays a binding site for p85α regulatory subunit of PI3K. By using murine primary microglial cultures, we show the presence of all CLM members in microglial cells and characterize the expression of CLM-1 both in basal conditions and during microglial activation. The TLR4 agonist lipopolysaccharide (LPS) and the TLR3 agonist polyinosinic-polycytidylic acid (Poly I:C) induce an increase in microglial CLM-1 mRNA levels in vitro, whereas the TLR2/6 heterodimer agonist peptidoglycan (PGN) produces a marked decrease. In this study we also describe a new soluble isoform of CLM-1 that is detected at mRNA and protein levels in basal conditions in primary microglial cultures. Interestingly, CLM-1 engagement enhances the transcription of the pro-inflammatory mediators TNFα, COX-2 and NOS-2 in microglial cells challenged with LPS. These results reveal that CLM-1 can acts as a co-activating receptor and suggest that this receptor could play a key role in the regulation of microglial activation

CD200R1 contributes to successful functional reinnervation after a sciatic nerve injury

Activating and inhibitory immune receptors play a critical role in regulating systemic and central nervous system (CNS) immune and inflammatory processes. The CD200R1 immunoreceptor induces a restraining signal modulating inflammation and phagocytosis in the CNS under different inflammatory conditions. However, it remains unknown whether CD200R1 has a role in modulating the inflammatory response after a peripheral nerve injury, an essential component of the successful regeneration. Expression of CD200R1 and its ligand CD200 was analyzed during homeostasis and after a sciatic nerve crush injury in C57Bl/6 mice. The role of CD200R1 inWallerian Degeneration (WD) and nerve regeneration was studied using a specific antibody against CD200R1 injected into the nerve at the time of injury. We found an upregulation of CD200R1 mRNA after injury whereas CD200 was downregulated acutely after nerve injury. Blockade of CD200R1 significantly reduced the acute entrance of both neutrophils and monocytes from blood after nerve injury. When long term regeneration and functional recovery were evaluated, we found that blockade of CD200R1 had a significant effect impairing the spontaneous functional recovery. Taken together, these results show that CD200R1 has a role in mounting a successful acute inflammatory reaction after injury, and contributes to an effective functional recovery

CD300f immunoreceptor contributes to peripheral nerve regeneration by the modulation of macrophage inflammatory phenotype

Background: It has recently become evident that activating/inhibitory cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). The immunoreceptor CD300f expressed on monocytes, neutrophils, and mast cells modulates inflammation, phagocytosis, and outcome in models of autoimmune demyelination, allergy, and systemic lupus erythematosus. On the other hand, a finely regulated inflammatory response is essential to induce regeneration after injury to peripheral nerves since hematogenous macrophages, together with resident macrophages and de-differentiated Schwann cells, phagocyte distal axonal and myelin debris in a well-orchestrated inflammatory response. The possible roles and expression of CD300f and its ligands have not been reported under these conditions. Methods: By using quantitative PCR (QPCR) and CD300f-IgG2a fusion protein, we show the expression of CD300f and its ligands in the normal and crush injured sciatic nerve. The putative role of CD300f in peripheral nerve regeneration was analyzed by blocking receptor-ligand interaction with the same CD300f-IgG2a soluble receptor fusion protein in sciatic nerves of Thy1-YFP-H mice injected at the time of injury. Macrophage M1/M2 polarization phenotype was also analyzed by CD206 and iNOS expression. Results: We found an upregulation of CD300f mRNA and protein expression after injury. Moreover, the ligands are present in restricted membrane patches of Schwann cells, which remain stable after the lesion. The lesioned sciatic nerves of Thy1-YFP-H mice injected with a single dose of CD300f-IgG2a show long lasting effects on nerve regeneration characterized by a lower number of YFP-positive fibres growing into the tibial nerve after 10 days post lesion (dpl) and a delayed functional recovery when compared to PBS- or IgG2a-administered control groups. Animals treated with CD300f-IgG2a show at 10 dpl higher numbers of macrophages and CD206-positive cells and lower levels of iNOS expression than both control groups. At later time points (28 dpl), increased numbers of macrophages and iNOS expression occur. Conclusions: Taken together, these results show that the pair CD300f ligand is implicated in Wallerian degeneration and nerve regeneration by modulating both the influx and phenotype of macrophages

Cu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury

BACKGROUND: In the nervous system, as in other organs, Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key antioxidant enzyme involved in superoxide detoxification in normal cellular metabolism and after cell injury. Although it has been suggested that immature brain has a different susceptibility to oxidative damage than adult brain, the distribution and cell-specific expression of this enzyme in immature brain and after postnatal brain damage has not been documented. METHODS: In this study, we used immunohistochemistry and western blot to analyze the expression of Cu/Zn SOD in intact immature rat brain and in immature rat brain after an NMDA-induced excitotoxic cortical injury performed at postnatal day 9. Double immunofluorescence labelling was used to identify Cu/Zn SOD-expressing cell populations. RESULTS: In intact immature brain, Cu/Zn SOD enzyme was widely expressed at high levels in neurons mainly located in cortical layers II, III and V, in the sub-plate, in the pyriform cortex, in the hippocampus, and in the hypothalamus. Glial fibrillary acidic protein-positive cells only showed Cu/Zn SOD expression in the glia limitans and in scattered cells of the ventricle walls. No expression was detected in interfascicular oligodendroglia, microglia or endothelial cells. Following excitotoxic damage, neuronal Cu/Zn SOD was rapidly downregulated (over 2–4 hours) at the injection site before neurodegeneration signals and TUNEL staining were observed. Later, from 1 day post-lesion onward, an upregulation of Cu/Zn SOD was found due to increased expression in astroglia. A further increase was observed at 3, 5 and 7 days that corresponded to extensive induction of Cu/Zn SOD in highly reactive astrocytes and in the astroglial scar. CONCLUSION: We show here that, in the intact immature brain, the expression of Cu/Zn SOD was mainly found in neurons. When damage occurs, a strong and very rapid downregulation of this enzyme precedes neuronal degeneration, and is followed by an upregulation of Cu/Zn SOD in astroglial cells

Sex-dependent role of CD300f immune receptor in generalized anxiety disorder.

Generalized Anxiety Disorder (GAD) presents a high prevalence in the population, leading to distress and disability. Immune system alterations have been associated with anxiety-related behaviors in rodents and GAD patients. CD300f immune receptors are highly expressed in microglia and participate not only in the modulation of immune responses but also in pruning and reshaping synapses. It was recently demonstrated that CD300f might be influential in the pathogenesis of depression in a sex-dependent manner. Here, we evaluated the role of CD300f immune receptor in anxiety, using CD300f knockout mice (CD300f-/-) and patients with GAD. We observed that male CD300f-/- mice had numerous behavioral changes associated with a low-anxiety phenotype, including increased open field central locomotion and rearing behaviors, more exploration in the open arms of the elevated plus-maze test, and decreased latency to eat in the novelty suppressed feeding test. In a cross-sectional population-based study, including 1111 subjects, we evaluated a common single-nucleotide polymorphism rs2034310 (C/T) in the cytoplasmatic tail of CD300f gene in individuals with GAD. Notably, we observed that the T allele of the rs2034310 polymorphism conferred protection against GAD in men, even after adjusting for confounding variables. Overall, our data demonstrate that CD300f immune receptors are involved in the modulation of pathological anxiety behaviors in a sex-dependent manner. The biological basis of these sex differences is still poorly understood, but it may provide significant clues regarding the neuropathophysiological mechanisms of GAD and can pave the way for future specific pharmacological interventions

Nanoparticulate architecture of protein-based artificial viruses is supported by protein DNA interactions

Aim & Methods: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine–glycine–aspartic acid cell-binding domains and promote significant transgene expression levels. Results & Conclusion: The structural analysis of the protein in these hybrid nanoparticles indicates a molecular conformation with predominance of D-helix and the absence of cross-molecular, E-sheet-supported protein interactions. The nanoscale organizing forces generated by DNA–protein interactions can then be observed as a potentially tunable, critical factor in the design of protein-only based artificial viruses for gene therapy.Agencia Nacional de Investigación e Innovació