Desenvolupament de proteïnes quimèriques multifuncionals com a vectors de transferència gènica mediada per receptor

Consultable des del TDXTítol obtingut de la portada digitalitzadaEls últims avenços en biologia molecular i cel·lular no tan sols han contribuït al coneixement de la base molecular de moltes malalties, sinó que també han proporcionat una tecnologia amb potencial de manipulació de gens in vivo. La teràpia gènica sorgeix com una estratègia terapèutica basada en la transferència de material genètic per al tractament de malalties d'origen genètic o infecciós, substituint, reparant o potenciant la funció biològica dels teixits o sistemes lesionats. Els principals estudis en aquesta línia d'investigació estan dirigits al desenvolupament de vectors eficients en la transferència gènica. En els últims anys, l'ús de vectors no vírics en teràpia gènica ha anat prenent importància, ja que tot i que s'ha demostrat que els vectors vírics són eines molt eficients en la transferència de DNA, presenten limitacions associades, com ara la difícil obtenció de títols elevats de partícules víriques, la inducció de respostes immunològiques i riscs de seguretat biològica com la mutagènesi insercional i la possible reversió a vectors recombinants competents en replicació. És molt important assegurar dins del ús de sistemes no vírics, processos de transferència gènica mediada per receptor, no tan sols per dirigir el procés d'internalització cel·lular eficientment, sinó per poder mantenir l'especificitat per un tipus cel·lular diana. Les proteïnes quimèriques recombinants són candidats molt interessants en la transferència gènica no vírica. La generació d'aquests vehicles ha estat poc explorada dins la teràpia gènica, i es basa en la combinació de diverses proteïnes o dominis proteics bioactius en una sola cadena polipeptídica. Aquestes regions heteròlogues combinades, aporten capacitat d'unir molècules de DNA, el reconeixement i la internalització cel·lular i possibles funcions potenciadores de la transferència cel·lular com ara la llisi d'endosomes i el transport nuclear. Els senzills processos de producció i purificació a gran escala i la seva naturalesa modular, fan que siguin vehicles fàcilment optimitzables i adequables a noves aplicacions de teràpia gènica i amb un elevat potencial com a vectors alternatius als sistemes vírics. En aquesta Tesi Doctoral s'han desenvolupat un grup de proteïnes quimèriques prototip, basades en l'enzim b-galactosidasa d'Escherichia coli, que presenten en la seva superfície una cua de lisines com a domini d'unió a DNA i la regió de reconeixement cel·lular del virus de la febre aftosa basada en motius RGD. A més a més, l'enzim b-galactosidasa funciona com a domini de purificació del constructe resultant i permet detectar i quantificar les proteïnes quimèriques mitjançant assaigs colorimètrics senzills. La presència de forma natural en aquest enzim d'una seqüència de localització nuclear críptica permet a aquestes proteïnes dirigir eficientment la transferència de DNA en cèl·lules de mamífer. La primera construcció obtinguda va ser anomenada 249AL i es va demostrar que unia i condensava DNA eficientment i era capaç de dirigir específicament, mitjançant el reconeixement de la integrina avb3, la seva transferència en cultius cel·lulars. Després d'optimitzar el procés de transferència gènica in vitro es van realitzar aproximacions in vivo injectant complexes de proteïna-DNA intracerebralment en rates. Els resultats mostraven que la proteïna 249AL és un prototip de vector alternatiu al ús de vectors vírics en el SNC, especialment en aplicacions terapèutiques a zones lesionades en les que es produeix un increment de l'expressió dels gens de les integrines avb3. Derivats de la proteïna 249AL, com la proteïna NLSCt, demostren que mitjançant la incorporació de nous dominis com ara altres senyals de localització nuclear, com la del Antigen-T de SV40, és poden obtenir nivells d'expressió gènica més elevats. Aquests resultats conviden a aprofundir en l'aproximació modular al disseny de vectors recombinants no vírics per a teràpia gènica.Recent advances in molecular and cellular biology have contributed to the knowledge of the molecular bases of some diseases and have provided new technologies for gene manipulation in vivo. Thus, gene therapy arises as a therapeutic strategy for gene delivery applied to the genetic and infectious diseases treatment, replacing, repairing or stimulating the biological function of affected tissues. The main studies in gene therapy have focused on the development of gene transfer vectors to ensure the efficiency in gene delivery processes. Some engineered viruses have been largely explored as such transfer vehicles with an important degree of success. However, a set of adverse reactions such as the difficult production of high viral particles titres, the induction of immune response and the associated biological risks such as insertional mutagenesis and the generation of replication competent viral particles, have strongly encouraged the use of non-viral vehicles for gene transfer. In the context of non-viral vectors, it is important to maintain receptor mediated gene transfer processes to ensure an efficient cellular internalisation and specificity. Chimerical recombinant proteins are interesting candidates in non-viral gene transfer and they have been constructed by combining bioactive proteins, or proteins domains, from different origins in a single polipeptidic chain. These joined regions supply DNA-condensing, cell-binding, internalisation and eventually endosome-disrupting and nuclear targeting activities. Although this family of constructs is still in an early stage of development, its intrinsic flexibility, the simplicity in the production and purification processes and the possibility of further improvement by protein engineering offers wider perspectives in gene therapy. In this Thesis it has been developed a new set of prototype chimerical proteins based on Escherichia coli b-galactosidase enzyme carrying a polylisine-based DNA binding domain and an integrin-targeting RGD cell binding peptide from Foot and Mouth Disease Virus (FMDV). The b-galactosidase acts as a purification domain of the final construct and permits its detection and quantification by simple colorimetric assays. The natural presence in this enzyme of a cryptic nuclear localisation signal (NLS) contributes to the efficiency of gene transfer to mammalian cells. The protein 249AL was the first construction obtained and it showed DNA binding and condensing abilities and led cellular gene transfer through the recognition of avb3 cellular integrins. After in vitro optimisation of gene delivery processes, in vivo approaches were carried out by injecting P9 rats intracerebrally with DNA-protein complexes. The results showed that 249AL protein is a promising alternative to viral vectors for CNS gene therapy, mainly in injured areas where the avb3 gene expression is increased. The 249AL derivatives, such as NLSCt protein, demonstrate that the incorporation of new functional domains such as other NLS, like that from SV40 T-antigen, could improve the obtained gene expression levels and invites explore in more detail this modular approach for the design of non-viral vectors in gene therapy

Design strategies for positively charged endolysins : Insights into Artilysin development

Altres ajuts: acords transformatius de la UABEndolysins are bacteriophage-encoded enzymes that can specifically degrade the peptidoglycan layer of bacterial cell wall, making them an attractive tool for the development of novel antibacterial agents. The use of genetic engineering techniques for the production and modification of endolysins offers the opportunity to customize their properties and activity against specific bacterial targets, paving the way for the development of personalized therapies for bacterial infections. Gram-negative bacteria possess an outer membrane that can hinder the action of recombinantly produced endolysins. However, certain endolysins are capable of crossing the outer membrane by virtue of segments that share properties resembling those of cationic peptides. These regions increase the affinity of the endolysin towards the bacterial surface and assist in the permeabilization of the membrane. In order to improve the bactericidal effectiveness of endolysins, approaches have been implemented to increase their net charge, including the development of Artilysins containing positively charged amino acids at one end. At present, there are no specific guidelines outlining the steps for implementing these modifications. There is an ongoing debate surrounding the optimal location of positive charge, the need for a linker region, and the specific amino acid composition of peptides for modifying endolysins. The aim of this study is to provide clarity on these topics by analyzing and comparing the most effective modifications found in previous literature

The chaperone DnaK controls the fractioning of functional protein between soluble and insoluble cell fractions in inclusion body-forming cells

Background: The molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities. We have here comparatively explored thermally promoted, in vivo protein aggregation and the formation of bacterial inclusion bodies, from both structural and functional sides. Also, the status of the soluble and insoluble protein versions in both aggregation systems have been examined as well as the role of the main molecular chaperones GroEL and DnaK in the conformational quality of the target polypeptide. Results: While thermal denaturation results in the formation of heterogeneous aggregates that are rather stable in composition, protein deposition as inclusion bodies renders homogenous but strongly evolving structures, which are progressively enriched in the main protein species while gaining native-like structure. Although both type of aggregates display common features, inclusion body formation but not thermal-induced aggregation involves deposition of functional polypeptides that confer biological activity to such particles, at expenses of the average conformational quality of the protein population remaining in the soluble cell fraction. In absence of DnaK, however, the activity and conformational nativeness of inclusion body proteins are dramatically impaired while the soluble protein version gains specific activity. Conclusion: The chaperone DnaK controls the fractioning of active protein between soluble and insoluble cell fractions in inclusion body-forming cells but not during thermally-driven protein aggregation. This cell protein, probably through diverse activities, is responsible for the occurrence and enrichment in inclusion bodies of native-like, functional polypeptides, that are much less represented in other kind of protein aggregates

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

Selecting subpopulations of high-quality protein conformers among conformational mixtures of recombinant bovine MMP-9 solubilized from inclusion bodies

Altres ajuts: CERCA Programme/Generalitat de CatalunyaA detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations

Potential of Oral Nanoparticles Containing Cytokines as Intestinal Mucosal Immunostimulants in Pigs : A Pilot Study

Antibiotics are essential compounds to cope with bacterial infections. However, their inadequate and excessive use has triggered the rapid arising of antimicrobial-resistant bacteria. In this scenario, immunostimulants, which are molecules that boost the immune system, open up a new approach to face this problem, enhancing treatment efficacy and preventing infections by immune system response. Cytokines are central effector molecules of the immune system, and their recombinant production and administration in animals could be an interesting immune modulation strategy. The aim of this study was the development of a highly stable nanoparticle of porcine cytokines to achieve the immunostimulation of intestinal mucosa in piglets. The outcomes of the present study prove this approach is able to stimulate swine intestinal cells and macrophages in vitro and tends to modulate inflammatory responses in vivo, although further studies are required to definitively evaluate their potential in animals. Antimicrobial resistance is a global threat that is worryingly rising in the livestock sector. Among the proposed strategies, immunostimulant development appears an interesting approach to increase animal resilience at critical production points. The use of nanoparticles based on cytokine aggregates, called inclusion bodies (IBs), has been demonstrated as a new source of immunostimulants in aquaculture. Aiming to go a step further, the objective of this study was to produce cytokine nanoparticles using a food-grade microorganism and to test their applicability to stimulate intestinal mucosa in swine. Four cytokines (IL-1β, IL-6, IL-8, and TNF-α) involved in inflammatory response were produced recombinantly in Lactococcus lactis in the form of protein nanoparticles (IBs). They were able to stimulate inflammatory responses in a porcine enterocyte cell line (IPEC-J2) and alveolar macrophages, maintaining high stability at low pH and high temperature. In addition, an in vivo assay was conducted involving 20 piglets housed individually as a preliminary exploration of the potential effects of IL-1β nanoparticles in piglet intestinal mucosa after a 7 d oral administration. The treated animals tended to have greater levels of TNF-α in the blood, indicating that the tested dose of nanoparticles tended to generate an inflammatory response in the animals. Whether this response is sufficient to increase animal resilience needs further evaluation

Potential of MMP-9 based nanoparticles at optimizing the cow dry period : pulling apart the effects of MMP-9 and nanoparticles

Altres ajuts: ICREA Academia awardThe cow dry period is a non-milking interval where the mammary gland involutes and regenerates to guarantee an optimal milk production in the subsequent lactation. Important bottlenecks such as the high risk of intramammary infections complicate the process. Antibiotics have been routinely used as a preventive treatment but the concerns about potential antibiotic resistance open a new scenario in which alternative strategies have to be developed. Matrix metalloproteinase-9 (MMP-9) is an enzyme able to degrade the extracellular matrix, triggering the involution and immune function of cow mammary gland. We have studied the infusion into the mammary gland of MMP-9 inclusion bodies as protein-based nanoparticles, demonstrating that 1.2 mg of MMP-9 enhanced the involution and immune function of the cow mammary gland. However, the comparison of the effects triggered by the administration of an active and an inactive form of MMP-9 led to conclude that the response observed in the bovine mammary gland was mainly due to the protein format but not to the biological activity of the MMP-9 embedded in the inclusion body. This study provides relevant information on the future use of protein inclusion bodies in cow mammary gland and the role of MMP-9 at dry-off

In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics

Oligomerization of antimicrobial peptides into nanosized supramolecular complexes produced in biological systems (inclusion bodies and self-assembling nanoparticles) seems an appealing alternative to conventional antibiotics. In this work, the antimicrobial peptide, GWH1, was N-terminally fused to two different scaffold proteins, namely, GFP and IFN-γ for its bacterial production in the form of such recombinant protein complexes. Protein self-assembling as regular soluble protein nanoparticles was achieved in the case of GWH1-GFP, while oligomerization into bacterial inclusion bodies was reached in both constructions. Among all these types of therapeutic proteins, protein nanoparticles of GWH1-GFP showed the highest bactericidal effect in an in vitro assay against Escherichia coli, whereas non-oligomerized GWH1-GFP and GWH1-IFN-γ only displayed a moderate bactericidal activity. These results indicate that the biological activity of GWH1 is specifically enhanced in the form of regular multi-display configurations. Those in vitro observations were fully validated against a bacterial infection using a mouse mastitis model, in which the GWH1-GFP soluble nanoparticles were able to effectively reduce bacterial loads

A new approach to obtain pure and active proteins from Lactococcus lactis protein aggregates

The production of pure and soluble proteins is a complex, protein-dependent and time-consuming process, in particular for those prone-to-aggregate and/or difficult-to-purify. Although Escherichia coli is widely used for protein production, recombinant products must be co-purified through costly processes to remove lipopolysaccharide (LPS) and minimize adverse effects in the target organism. Interestingly, Lactococcus lactis, which does not contain LPS, could be a promising alternative for the production of relevant proteins. However, to date, there is no universal strategy to produce and purify any recombinant protein, being still a protein-specific process. In this context and considering that L. lactis is also able to form functional protein aggregates under overproduction conditions, we explored the use of these aggregates as an alternative source of soluble proteins. In this study, we developed a widely applicable and economically affordable protocol to extract functional proteins from these nanoclusters. For that, two model proteins were used: mammary serum amyloid A3 (M-SAA3) and metalloproteinase 9 (MMP-9), a difficult-to-purify and a prone-to-aggregate protein, respectively. The results show that it is possible to obtain highly pure, soluble, LPS-free and active recombinant proteins from L. lactis aggregates through a cost-effective and simple protocol with special relevance for difficult-to-purify or highly aggregated proteins