Neuroinflammation and Gene Therapy
- Hugo Peluffo, PhD (Joint Position) Prof. Agregado (G4, Full-Time) Depto. Histología Y Embriología Facultad de Medicina, Universidad de la República (UDELAR)
Members at the IPMon
- Natalia Lago (PhD, Assistant Researcher, Part Time)
- Luciana Negro (PhD Student and Prof. G2 at the Depto. Histología Y Embriología Facultad de Medicina, Universidad de la República-UDELAR)
- Bruno Pannunzio (Student)
- Daniela Alí (MSc Student)
- Andrés Cawen (Student)
Members at the Faculty of Medicine
- Daniela Blanco (MSc Student)
- Nathalia Vitureira (PhD, Investigador Asociado IPMon)
Nervous System Traumatic Injuries
Traumatic injuries to the Nervous System, including traumatic brain injury (TBI) and spinal cord injury (SCI), remain one of the leading causes of mortality and morbidity in both industrialized and developing countries, being of increased importance in the latter. TBI is frequently referred to as the “silent epidemic”, as beyond symptoms like paralysis, additional complications such as changes affecting intellectual abilities, sensation, language, or emotions, may not be readily apparent. In fact, studies including several European countries showed that TBI resulted between the highest injury burden pathologies due to permanent disability, and among the highest costs for the health system. Extensive efforts have been made to reach neuroprotective therapies for these devastating disorders, but despite interesting preclinical results, no successful outcomes have been observed in human clinical trials to date. Following the initial mechanical insult, focal TBI and SCI results in a complex delayed secondary progressing injury due to anatomical, neurochemical, metabolic, inflammatory and cellular changes that account for many of the neurological deficits observed. Inflammatory and immune reactions are present in all acute and chronic neurological pathologies. Interestingly, these processes are not only a consequence of neurodegeneration but also a critical mediator of the neurotoxic or neuroprotective mechanisms. Thus their modulation has emerged as an important therapeutic opportunity.
Neuroinflammation and CNS Damage
Although the brain has long been considered an “immune-privileged” organ, this status is far from absolute. CNS cells have innate immune functions and express a range of receptors capable of detecting and clearing apoptotic cells and regulating inflammatory responses. Among them, bone marrow-derived MICROGLIAL CELLS, are the main nervous component of the innate immune system. Resident microglia survey the CNS and act as the first line of defense against pathogen invasion by recognizing, sequestering and processing antigens, but also participate in processes regarding neuronal communication and homeostasis. Acute lesions induce tissue damage and neurodegeneration which, in turn, incite an inflammatory response characterized by the activation of microglia, astrocytes, endothelial cells, blood leukocytes, in a process highly dependent on the type of injury and the degree of tissue damage. Several evidences have shown that the production of inflammatory molecules and oxidative stress by inflammatory cells determine the final extent of tissue damage and the death or survival of neuronal cells in surrounding or distally connected areas. Moreover, the inflammatory processes also contribute to the barriers for regeneration and plasticity.
Control of Inflammation
Inflammation is a set of complex interactions between soluble factors, extracellular matrix and cells, which is induced in any tissue in response to injury or infection. Inflammation in peripheral organs normally leads to tissue recovery, but if destruction of pathogens and resolution of damaged cells and matrix are not adequately controlled, inflammation can lead to persistent damage. For this reason, checkpoints for the control of inflammatory mechanisms, which are induced as a result of the activation of the inflammatory cascade, have gained a high degree of importance and interest in the field of immunology. Importantly, recent findings suggest that the anti-inflammatory state is not only a passive state resulting from an absence of inflammatory stimuli, but an active condition that requires participation of several molecules responsible for the supression of potentially inflammatory stimuli. This is one of the central hypothesis of our research group.
In this sense, regulation of immune cell function by inhibitory/regulatory receptors has been characterized in the immune system, and just recently few studies have attempted their participation in the regulation of microglial cell activation after acute CNS injury. Balance between the destructive/protective events of the innate response must be precisely regulated in order to limit initial toxicity and promote CNS repair and a return to homeostatic conditions. In the last few years, promising activating/inhibitory immune receptors have been highlighted as new targets for the control and modulation of microglia/macrophage responses include the CD200/CD200R system, TREM-2 receptor and the recently described family of CD300 receptors [1-5].
CD300 Family of Receptors
The human IREM/CD300 family of activating/inhibitory receptors is composed by six members, CD300a/IRP60, CD300b/IREM3, CD300c/CMRF35, CD300d, CD300e/IREM2 and CD300f/IREM1. The importance of this family of receptors is highlighted by the fact that CD300a is the second gene with strongest evidence for positive selection between human and chimpanzee. Moreover, CD300a and CD300f are among the 10 highest genes upregulated after rat SCI. All of the members share an extracellular region comprising a single Ig-like domain and, with the exception of CD300a, a myeloid linage restricted pattern of expression. Two of the activating members, CD300b and CD300e, fit to the classical scheme for activating receptors with a positive charge within their membrane domain. They recruite the transmembrane adaptor molecule DAP-12 through the positive charge in its transmembrane domain andhave a functional tyrosine residue in its cytoplasmic tail able to recruit Grb2,thus signalling through two different pathways. The CD300 family contains two inhibitory receptors, CD300a and CD300f. Both display a long cytoplasmic tail with a variety of different tyrosine-based motifs and both are able to recruit SHP-1 phosphatase and therefore deliver inhibitory signals.
The most interesting difference between these molecules, besides their different pattern of expression, is the existence of two binding motifs for the p85 subunit of PI3Kinase in the cytoplasmic tail of CD300f. In fact, it has been shown that CD300f delivers in vitro both inhibitory and activating signals, thus revealing a remarkable functional duality of this receptor, similar to what has been shown for TREM2, another dual activating receptor. However, in vivo CD300f has shown to be mainly an inhibitory receptor, as shown in CD300f knockout animals in the EAE model of Multiple Sclerosis, and very recently in several models of Allergyand in a model of Lupus Eritematosus.
It has been shown that the CD300 receptors are able to form complexes on the cell surface through the interaction among their extracellular immunoglobulin domain,and their combination in a complex differentially modulated the signalling outcome, suggesting how CD300 complexes could regulate the activation of myeloid cells upon interaction with their natural ligands. It was published that the T cell immunoglobulin mucin (TIM) proteins TIM-1 and TIM-4, which regulate T cell activation and tolerance, are ligands for the mouse CD300b receptor . However, very recent reports suggest the existence of othermain ligands for the CD300 receptors as the phospholipids phosphatidylserine, phosphatidylcholine or sphingomyelin and ceramide.
Innovative Gene Therapy Strategies For Traumatic Injury of The Nervous System
The introduction of functional genes into an organism, as well as the regulation of gene expression has emerged in the past few decades as a powerful tool for treating or correcting multiple pathologies. To date, over 2076 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. The first commercial gene therapy drug has been approved and there are several advanced clinical trials (75 in phase III and 2 in phase IV), showing the important development of this area. One of the main focuses in gene therapy has been the development of sophisticated delivery systems, which can constitute the bottleneck for the achievement of clinical effects. As virus are evolutionary optimized for this purpose, viral vectors tend to be the most effective carriers of nucleic acids into foreign cells. In particular, several vectors show promising features for the use in the nervous system including non-integrating lentiviral vectors and some serotypes of Adenoassociated viral vectors with blood-brain barrier crossing potential.
One of the most popular types of viral vectors for treating CNS disorders are HIV 1-derived lentiviral vectors. They have been tailored in the past years to reduce their biological risks and to display several features that make them excellent candidates for treating CNS disorders including low immunogenicity and transduction of post-mitotic cells. Non-viral vectors have also gained attention, and in particular, vehicles based on multifunctional proteins in DNA complexes constitute a very versatile type of carriers for therapeutic nucleic acids. They are constructed by the combination of appropriate functional domains fused in a single polypeptide chain. This approach has generated the first prototypes of modular recombinant protein nano-vectors where the integrated domains enable the whole construct to mimic the infective viral cycle, which is necessary to the targeted delivery of nucleic acids. Thus, this type of nanoparticles have been also termed artificial virus.
The modular nature of such constructs allows the selection of different features using well-characterized peptides and a functional redesign in iterative improvement processes. Several of these nano-vectors have shown successful transfection in vitro and therapeutic effects in vivo, suggesting their potential in the clinical context. One of these, the modular nano-vector termed NLSCt, is based on the tetramer carrier protein β-Galactosidase engineered with a polylysine K10 tail which bind and condense DNA, a NLS motif for nuclear localization and an prototypic integrin-interacting RGD domain which binds to membrane integrins and promotes cell internalization. We showed for the first time that these types of vectors induce biologically relevant concentrations of transgenic protein after acute excitotoxic brain injuries[5, 12-15]. Interestingly, the RGD interacting motif of the NLSCt protein was able to induce neuroprotection per se, enabling the possibility of directing rapid actions of the vectors through the selection of their functional motifs, constituting the proof-of-principle for a “trophic vector”. The modular principles underlying the NLSCt vector were further improved by generating two smaller nano-vectors termed HKRN and HNRK, based on alternative direct combination of the three functional domains RGD, NLS and K10 in a single small polypeptide, with the addition of a poly-histidine domain H6 that provides endosomal escape and purification properties. These nano-vectors achieved significant transgene expression levels in culture cells, and in vivo after a TBI .
Importantly, the original hypothesis that a very efficient vector could be used for most gene therapy application has evolved to the notion that each particular pathological condition may need a particular vector. For instance, for the treatment of acute traumatic CNS injuries, a gene therapy vector should induce a rapid but not permanent induction of transgene expression, it should not be proinflammatory as inflammation is a key mediator of the neuropathology, and a specific cell type may or may not needed to be targeted depending on the mechanism of action of the transgene. However other variables are less evident: i) which are the desired levels of transgene expression? ii) Which is the ideal time frame of expression? iii) Should the vector induce widespread or a localized transduction? The lack of detailed comparisons of different types of vectors in the same model under identical conditions hamper the selection of the best vector under these particular pathological conditions. This has an important impact on useful translational medicine approaches, were detailed comparative estudies are essential. In fact, this constitutes one of the main focuses of our research group.
- Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, Blom B, Homola ME, Streit WJ, Brown MH, et al: Down-regulation of the macrophage lineage through interaction with OX2 (CD200).Science 2000, 290:1768-1771.
- Kleinberger G, Yamanishi Y, Suarez-Calvet M, Czirr E, Lohmann E, Cuyvers E, Struyfs H, Pettkus N, Wenninger-Weinzierl A, Mazaheri F, et al: TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis.Sci Transl Med 2014, 6:243ra286.
- Neumann H, Takahashi K: Essential role of the microglial triggering receptor expressed on myeloid cells-2 (TREM2) for central nervous tissue immune homeostasis.J Neuroimmunol 2007, 184:92-99.
- Xi H, Katschke KJ, Jr., Helmy KY, Wark PA, Kljavin N, Clark H, Eastham-Anderson J, Shek T, Roose-Girma M, Ghilardi N, van Lookeren Campagne M: Negative regulation of autoimmune demyelination by the inhibitory receptor CLM-1.J Exp Med 2010, 207:7-16.
- Peluffo H, Ali-Ruiz D, Ejarque-Ortiz A, Heras-Alvarez V, Comas-Casellas E, Martinez-Barriocanal A, Kamaid A, Alvarez-Errico D, Negro ML, Lago N, et al: Overexpression of the immunoreceptor CD300f has a neuroprotective role in a model of acute brain injury.Brain Pathol 2011, 22:318-328.
- Borrego F: The CD300 molecules: an emerging family of regulators of the immune system.Blood.
- Nielsen R, Bustamante C, Clark AG, Glanowski S, Sackton TB, Hubisz MJ, Fledel-Alon A, Tanenbaum DM, Civello D, White TJ, et al: A scan for positively selected genes in the genomes of humans and chimpanzees.PLoS Biol 2005, 3:e170.
- Torres-Espin A, Hernandez J, Navarro X: Gene expression changes in the injured spinal cord following transplantation of mesenchymal stem cells or olfactory ensheathing cells.PLoS One 2013, 8:e76141.
- Izawa K, Yamanishi Y, Maehara A, Takahashi M, Isobe M, Ito S, Kaitani A, Matsukawa T, Matsuoka T, Nakahara F, et al: The receptor LMIR3 negatively regulates mast cell activation and allergic responses by binding to extracellular ceramide.Immunity 2012, 37:827-839.
- Tian L, Choi SC, Murakami Y, Allen J, Morse HC, 3rd, Qi CF, Krzewski K, Coligan JE: p85alpha recruitment by the CD300f phosphatidylserine receptor mediates apoptotic cell clearance required for autoimmunity suppression.Nat Commun 2014, 5:3146.
- Peluffo H: Modular Multifunctional Protein Vectors for Gene Therapy. In Non-Viral Gene Therapy. Edited by Yuan X: InTech; 2011: 597-614
- Peluffo H, Acarin L, Aris A, Gonzalez P, Villaverde A, Castellano B, Gonzalez B: Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector.BMC Neurosci 2006, 7:35.
- Peluffo H, Aris A, Acarin L, Gonzalez B, Villaverde A, Castellano B: Nonviral gene delivery to the central nervous system based on a novel integrin-targeting multifunctional protein.Hum Gene Ther 2003, 14:1215-1223.
- Gonzalez P, Peluffo H, Acarin L, Villaverde A, Gonzalez B, Castellano B: Interleukin-10 overexpression does not synergize with the neuroprotective action of RGD-containing vectors after postnatal brain excitotoxicity but modulates the main inflammatory cell responses.J Neurosci Res, 90:143-159.
- Peluffo H, Gonzalez P, Acarin L, Aris A, Beyaert R, Villaverde A, Gonzalez B: Overexpression of the nuclear factor kappaB inhibitor A20 is neurotoxic after an excitotoxic injury to the immature rat brain.Neurol Res 2013, 35:308-319.
- Peluffo H, Gonzalez P, Aris A, Acarin L, Saura J, Villaverde A, Castellano B, Gonzalez B: RGD domains neuroprotect the immature brain by a glial-dependent mechanism.Ann Neurol 2007, 62:251-261.
- Negro-Demontel ML, Saccardo P, Giacomini C, Yáñez-Muñoz RJ, Ferrer-Miralles N, Vazquez E, Villaverde A, Peluffo H: Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy.Molecular Therapy — Methods & Clinical Development 2014, 1:14047.
- Regional Course&Symposium: “Neuron Glia Interactions in health and disease: from basic Biology to translationalNeuroscience – 2nd edition” 29 september-2 october, 2014.
- Project FundacióMarató TV3, Catalunya, España. “Modulation of immune receptors function as a novel therapeutic strategy for acute CNS damage”. (2012-2014) Amount Granted € 80.000. View results obtained here
- Project CSIC-UDELAR: “Gene therapy applied to brain trauma: comparative preclinical studies using modular recombinant vectors and lentiviral vectors”. (2013-2014) Amount granted USD 40.000.
- Project CSIC-UDELAR Grupos I+D: Neuroinflammation and glia”. (2015-2016) Coordinated project.
PUBLICATIONS (last 5 years)
- Domingo-Espín, J., E. Vazquez, J. Ganz, O. Conchillo, E. García-Fruitós, J. Cedano, U. Unzueta, V. Petegnief, N. Gonzalez-Montalbán, A.M. Planas, X. Daura, H. Peluffo, N. Ferrer-Miralles, y A. Villaverde. The nanoparticulate architecture of protein-based artificial viruses is supported by protein-DNA interactions. Nanomedicine. 6:1047-1061 (2011).
- Peluffo, Modular Multifunctional Protein Vectors for Gene Therapy. En: Non-viral Gene Therapy, Prof. Xubo Yuan Ed., Editorial INTECH, 2011, ISBN: 9789533075389.
- Fricker FR, Lago N, Balarajah S, Tsantoulas C, Tanna S, Zhu N, Fageiry SK, Jenkins MG, Garratt A, Birchmeier C, Bennett DLH. Axonally derived Neuregulin-1 is required for remyelination and regeneration following nerve injury in adulthood. Journal of Neuroscience, 2011; 31:3225-33.
- Pau Gonzalez; Hugo Peluffo; Laia Acarin; Antonio Villaverde; Berta Gonzalez y Bernardo Castellano. IL-10 overexpression does not synergize with the neuroprotective action of RGD-containing vectors after postnatal brain excitotoxicity, but modulates the main inflammatory cell responses. Journal of Neuroscience Research, (Epub 2011) 90:143-59 (2012).
- Peluffo, H*; Alí-Ruiz, D; Ejarque-Ortíz, A; Heras-Alvarez, V; Comas-Casellas, E; Martínez-Barriocanal, A; Kamaid, A; Alvarez-Errico, D; Negro, ML; Lago, N; Schwartz S Jr; Villaverde, A; y Sayós, J. Overexpression of the immunoreceptor CD300f has a neuroprotective role in a model of acute brain injury. Brain Pathology (Epub 2011) 22:318-328 (2012). * Autor al que debe dirigirse la correspondencia.
- Fitzgerald JJ, Lago N, Benmerah S, Serra J, Watling CP, Cameron RE, Tarte E, Lacour SP, McMahon SB, Fawcett JW. A regenerative microchannel neural interface for recording from and stimulating peripheral axons in vivo. J Neural Eng 2012; Feb 9.
- *Peluffo, H., *Foster, E., Ahmed, S.G., Lago, N., Hutson, T.H., Moon, L., Yip, P., Wanisch, K., Caraballo-Miralles, V., Olmos, G., Lladó, J., McMahon, S.B. and Yáñez-Muñoz, R.J. Efficient gene expression from integration-deficient lentiviral vectors in the spinal cord. Gene Therapy 2012 doi: 10.1038/gt.2012.78. [Epub ahead of print]. *Ambos autores contribuyeron igualmente al trabajo.
- Joan Domingo-Espín, Valérie Petegnief, Núria de Vera, Oscar Conchillo-Solé, Paolo Saccardo, Ugutz Unzueta, Esther Vazquez, Juan Cedano, Luciana Negro, Xavier Daura, Hugo Peluffo, Anna M. Planas, Antonio Villaverde, Neus Ferrer-Miralles. RGD-based cell ligands for cell-targeted drug delivery act as potent trophic factors. Nanomedicine: Nanotechnology, Biology and Medicine. 2012 Nov;8(8):1263. (Epub 2012 Jul 25)
- Hugo Peluffo*, Pau Gonzalez, Laia Acarin, Anna Aris, Rudy Beyaert, Antonio Villaverde y Berta Gonzalez. Overexpression of the nuclear factor kappa B inhibitor A20 is neurotoxic after an excitotoxic injury to the immature rat brain. Neurological Research 35(3):308-319, 2013. *Autor al que debe dirigirse la correspondencia.
- Negro, M.L. P. Saccardo, C. Giacomini, R.J. Yáñez-Muñoz, N. Ferrer-Miralles, E. Vazquez, A. Villaverde and H, Peluffo*. Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy. Molecular Therapy – Methods & Clinical Development, 1:14047, 2014. *Autor al que debe dirigirse la correspondencia.
- Lago N, Quintana A, Carrasco J, Giralt M, Hidalgo J, Molinero A. Absence of metallothionein-3 produces changes on MT-1/2 regulation in basal conditions and alters hypothalamic-pituitary-adrenal (HPA) axis. Neurochem Int. 74:65-73, 2014
- Aroa Ejarque-Ortiz, Carme Solà, Águeda Martínez-Barriocanal, Simó Schwartz Jr., Margarita Martín, Hugo Peluffo, Joan Sayós. The receptor cmrf35-like molecule-1 (clm-1) enhances the production of LPS-induced pro-inflammatory mediators during microglial activation. PLoS ONE DOI: 10.1371/journal.pone.0123928, 2015.
- Peluffo H, Solari-Saquieres P, Negro-Demontel ML, Isaac Francos-Quijorna, Navarro X, Ruben López-Vales, Sayós J, Lago N. CD300f immunoreceptor contributes to peripheral nerve regeneration by the modulation of macrophage inflammatory phenotype. J. Neuroinflammation, 12:145 (12 August) 2015.
- Hugo Peluffo, Ugutz Unzueta, María Luciana Negro, Zhikun Xu, Esther Vazquez, Neus Ferrer-Miralles and Antonio Villaverde. BBB-targeting, protein-based nanomedicines for drug and nucleic acid delivery to the CNS. Biotechnology Advances, 33(2):277-287, 2015.
- Santos-Nogueira, López-Serrano, Hernández, Lago N, Astudillo AM, Balsinde J, Estivill-Torrús G, de Fonseca FR, Chun J, López-Vales R. Activation of Lysophosphatidic Acid Receptor Type 1 Contributes to Pathophysiology of Spinal Cord Injury. Journal of Neuroscience, 4703-14, 2015.