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

Citrus aurantium flavonoid extract improves concentrate efficiency, animal behavior, and reduces rumen inflammation of Holstein bulls fed high-concentrate diets

One hundred fourty-four bulls (164.8 ± 5.91 kg BW and 135 ± 7.2 d of age) were randomly allocated to one of 8 pens and assigned to control (C) or citrus flavonoid (BF) treatments (Citrus aurantium, 0.4 kg per ton of concentrate of Bioflavex CA, > 20% naringin; BF). Each pen had one drinker, one separate five-space straw feeder, and one separate three-space feeder where mash concentrate containing mostly corn, barley, DDG and wheat was offered. Concentrate intake was recorded daily, whilst BW and animal behavior were recorded fortnightly. Animals were slaughtered after 168 d of study (12 periods of 14 d), and HCW and carcass quality were recorded, and rumen papillae samples were collected. Final BW (437.9 ± 1.85 kg), HCW (238.7 ± 2.02 kg), and concentrate intake (7.1 ± 0.13 kg/d) were not affected by treatment. Concentrate feed conversion ratio (kg of concentrate/ kg of BW) tended (P < 0.10) to be lesser in BF than in C bulls (5.11 vs. 5.36 ± 0.108 kg/kg). Percentage of animals eating concentrate during visual scan was greater (P < 0.01) in BF compared with C bulls (10.02% vs. 7.97% ± 0.512). Oral non-nutritive behaviors, agonistic interactions (fighting, butting, and chasing) and sexual behaviors (flehmen, attempted and complete mounts) were greater (P < 0.01) in C than in BF bulls. In the rumen epithelium, gene expression of bitter taste receptor 7, bitter taste receptor 16, bitter taste receptor 38 and bitter taste receptor 39 was greater (P < 0.05) in C compared with BF bulls, as well as was gene expression of free fatty acid receptor 2, pancreatic polypeptide receptor 1, cholecystokinin receptor 4, cytokine IL-25, Toll-like receptor-4 and β-defensin1. In conclusion, supplementation with flavonoids extracted from Citrus aurantium in bulls fed high-concentrate diets tends to improve efficiency, and reduces oral non-nutritive behaviors, agonistic interactions and sexual behavior. Moreover, flavonoid supplementation modifies the expression of genes in the rumen epithelium that could be related with eating and animal behavior regulation.info:eu-repo/semantics/acceptedVersio

Effects of Flavonoids Extracted from Citrus aurantium on Performance, Behavior, and Rumen Gene Expression in Holstein Bulls Fed with High-Concentrate Diets in Pellet Form

Flavonoid supplementation may modify the behavior and rumen inflammatory response of fattening bulls, and this could be related to the concentrate presentation (mash or pellet) form. In the present study, 150 Holstein bulls (183.0 ± 7.53 kg BW and 137 ± 1.8 d of age) were randomly allocated to one of eight pens and assigned to control (C) or (BF) (Citrus aurantium, Bioflavex CA, HealthTech Bio Actives, Spain, 0.4 kg per ton of concentrate of Bioflavex CA, 20% naringin). Concentrate (pellet) intake was recorded daily, and BW and animal behavior fortnightly. Animals were slaughtered after 168 d of study, and ruminal epithelium samples were collected for gene expression analyses. Treatment did not affect animal performance; however, BF supplementation reduced agonistic interactions and oral non-nutritive behaviors and increased the time devoted to eating concentrate and ruminating activity (p < 0.05). The gene expression of some genes in the rumen epithelium was greater or tended to be greater in BF than C bulls (bitter taste receptor 16, cytokine IL-25, β-defensin; p < 0.10; pancreatic polypeptide receptor 1 and tumor necrosis factor alpha; p < 0.05). In conclusion, flavonoid supplementation modifies the expression of genes in the rumen epithelium that could be related to inflammation and animal behavior modulation.info:eu-repo/semantics/publishedVersio

Short Communication: The biological value of transition milk: analyses of Immunoglobulin G, IGF-I and Lactoferrin in primiparous and multiparous dairy cows

Colostrum (the first mammary gland secretion after calving) is known to contain high concentrations of nutrients as well as bioactive substances (including immunoglobulins, growth factors, and antimicrobial factors) to ensure neonatal survival. Due to its immunomodulatory, antibacterial, and antiviral activities, bovine colostrum has been used not only in calves but also in the prevention and treatment of human gastrointestinal and respiratory infections. Transition milk is the mammary secretion from the second milking to the sixth, which may contain these bioactive compounds to a lesser extent. The objective of the present study was to measure IGF-I, immunoglobulin G (IgG), and lactoferrin (LTF) concentrations in colostrum and transition milk of primiparous and multiparous cows to further assess its potential use in veterinary and nutraceutical applications. The results demonstrated that the concentrations of these three bioactive molecules decrease from the first milking to the tenth. Concentrations of IGF-I and LTF were greater in multiparous than in primiparous cows. Also, lactation number interacted with milking number in IGF-I, since primiparous cows had a smoother decline of IGF-I concentrations than multiparous ones. Overall, transition milk from the second milking showed a 46% decrease in the analysed colostrum bioactive molecules. Therefore, further studies are needed to apply this knowledge in neonate farm management practices or in developing pharmaceutical supplements from farm surpluses.info:eu-repo/semantics/publishedVersio

Insertional protein engineering for analytical molecular sensing

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors

The future of recombinant host defense peptides

The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.info:eu-repo/semantics/publishedVersio

Trends in recombinant protein use in animal production

Recombinant technologies have made possible the production of a broad catalogue of proteins of interest, including those used for animal production. The most widely studied proteins for the animal sector are those with an important role in reproduction, feed efficiency, and health. Nowadays, mammalian cells and fungi are the preferred choice for recombinant production of hormones for reproductive purposes and fibrolytic enzymes to enhance animal performance, respectively. However, the development of low-cost products is a priority, particularly in livestock. The study of cell factories such as yeast and bacteria has notably increased in the last decades to make the new developed reproductive hormones and fibrolytic enzymes a real alternative to the marketed ones. Important efforts have also been invested to developing new recombinant strategies for prevention and therapy, including passive immunization and modulation of the immune system. This offers the possibility to reduce the use of antibiotics by controlling physiological processes and improve the efficacy of preventing infections. Thus, nowadays different recombinant fibrolytic enzymes, hormones, and therapeutic molecules with optimized properties have been successfully roduced through cost-effective processes using microbial cell factories. However, despite the important achievements for reducing protein production expenses, alternative strategies to further reduce these costs are still required. In this context, it is necessary to make a giant leap towards the use of novel strategies, such as nanotechnology, that combined with recombinant technology would make recombinant molecules affordable for animal industry.info:eu-repo/semantics/publishedVersio

Design strategies for positively charged endolysins: Insights into Artilysin development

Endolysins 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 authors are indebted to Agencia Española de Investigación for the granted project (PID2019-107298RB-C21/AEI/10.13039/501100011033 to EGF and AA and PID2019-107298RB-C22/AEI/10.13039/501100011033 to NFM), to Marató de TV3 foundation (grant 201812-30-31-32-33) and to AGAUR for project 2021 SGR 01552. The authors are also indebted to the CERCA Program (Generalitat de Catalunya) and European Social Fund for supporting our research. JVCT is supported with a Margarita Salas grant for the training of young doctoral graduates (722713).info:eu-repo/semantics/publishedVersio

A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity

Background Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity. Results A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM. Conclusions Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.info:eu-repo/semantics/publishedVersio