Chitosan-Based Drug Delivery System: Applications in Fish Biotechnology

Chitosan is increasingly used for safe nucleic acid delivery in gene therapy studies, due to well-known properties such as bioadhesion, low toxicity, biodegradability and biocompatibility. Furthermore, chitosan derivatization can be easily performed to improve the solubility and stability of chitosan-nucleic acid polyplexes, and enhance e cient target cell drug delivery, cell uptake, intracellular endosomal escape, unpacking and nuclear import of expression plasmids. As in other fields, chitosan is a promising drug delivery vector with great potential for the fish farming industry. This review highlights state-of-the-art assays using chitosan-based methodologies for delivering nucleic acids into cells, and focuses attention on recent advances in chitosan-mediated gene delivery for fish biotechnology applications. The e ciency of chitosan for gene therapy studies in fish biotechnology is discussed in fields such as fish vaccination against bacterial and viral infection, control of gonadal development and gene overexpression and silencing for overcoming metabolic limitations, such as dependence on protein-rich diets and the low glucose tolerance of farmed fish. Finally, challenges and perspectives on the future developments of chitosan-based gene delivery in fish are also discussed

Gene markers of dietary macronutrient composition and growth in the skeletal muscle of gilthead sea bream (Sparus aurata)

To increase our current knowledge on the nutritional regulation of growth and gene expression pattern in fish skeletal muscle, the effect of dietary macronutrient composition was assessed on digestibility, nutrient retention, growth performance, and the mRNA levels of key genes involved in functionality, growth and development of the skeletal muscle in gilthead sea bream (Sparus aurata). Long-term starvation decreased the expression of myogenic regulatory factors such as Myod2, Myf5, myogenin (Myog) and Myf6 in the skeletal muscle of S. aurata. The supply of high or medium protein, low carbohydrate diets enhanced growth parameters, feed efficiency ratio, feed conversion ratio and significantly upregulated myod2. However, the supply of low protein, high carbohydrate diets restricted growth and stimulated the mRNA levels of myostatin, while downregulated follistatin (fst), igf1, mtor and rps6. Microarray analysis revealed igfals, tnni2, and gadd45a as gene markers upregulated by diets enriched with protein, lipids and carbohydrates, respectively. The results of the present study show that in addition to myod2, fst, igf1, mtor and rps6, the expression levels of igfals, tnni2 and remarkably gadd45a in the skeletal muscle can be used as markers to evaluate the effect of dietary macronutrient changes on fish growth and muscle development in S. aurata.This work was supported by the Ministerio de Economía, Industria y Competitividad, Spain (grant no. AGL2016-78124-R; cofunded by the European Regional Development Fund, European Commission) and the Agencia Nacional de Investigacion ´ y Desarrollo, Chile (Becas Chile/ 2011–72111506). The authors thank Piscicultura Marina Mediterranea (Burriana, Castellon, ´ Spain) for providing S. aurata juveniles.Peer ReviewedPostprint (published version

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons

Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cβ2β3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 μM. Interestingly, in the presence of 30 μM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine’s EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both α6N/α3Cβ2β3-nAChRs and α6M211L/α3ICβ2β3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cβ2β3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence

Gene markers of dietary macronutrient composition and growth in the skeletal muscle of gilthead sea bream (Sparus aurata)

To increase our current knowledge on the nutritional regulation of growth and gene expression pattern in fish skeletal muscle, the effect of dietary macronutrient composition was assessed on digestibility, nutrient retention, growth performance, and the mRNA levels of key genes involved in functionality, growth and development of the skeletal muscle in gilthead sea bream (Sparus aurata). Long-term starvation decreased the expression of myogenic regulatory factors such as Myod2, Myf5, myogenin (Myog) and Myf6 in the skeletal muscle of S. aurata. The supply of high or medium protein, low carbohydrate diets enhanced growth parameters, feed efficiency ratio, feed conversion ratio and significantly upregulated myod2. However, the supply of low protein, high carbohydrate diets restricted growth and stimulated the mRNA levels of myostatin, while downregulated follistatin (fst), igf1, mtor and rps6. Microarray analysis revealed igfals, tnni2, and gadd45a as gene markers upregulated by diets enriched with protein, lipids and carbohydrates, respectively. The results of the present study show that in addition to myod2, fst, igf1, mtor and rps6, the expression levels of igfals, tnni2 and remarkably gadd45a in the skeletal muscle can be used as markers to evaluate the effect of dietary macronutrient changes on fish growth and muscle development in S. aurata

The VCAM1-ApoE pathway directs microglial chemotaxis and alleviates Alzheimer\u27s disease pathology

In Alzheimer\u27s disease (AD), sensome receptor dysfunction impairs microglial danger-associated molecular pattern (DAMP) clearance and exacerbates disease pathology. Although extrinsic signals, including interleukin-33 (IL-33), can restore microglial DAMP clearance, it remains largely unclear how the sensome receptor is regulated and interacts with DAMP during phagocytic clearance. Here, we show that IL-33 induces VCAM1 in microglia, which promotes microglial chemotaxis toward amyloid-beta (Aβ) plaque-associated ApoE, and leads to Aβ clearance. We show that IL-33 stimulates a chemotactic state in microglia, characterized by Aβ-directed migration. Functional screening identified that VCAM1 directs microglial Aβ chemotaxis by sensing Aβ plaque-associated ApoE. Moreover, we found that disrupting VCAM1-ApoE interaction abolishes microglial Aβ chemotaxis, resulting in decreased microglial clearance of Aβ. In patients with AD, higher cerebrospinal fluid levels of soluble VCAM1 were correlated with impaired microglial Aβ chemotaxis. Together, our findings demonstrate that promoting VCAM1-ApoE-dependent microglial functions ameliorates AD pathology

An \u3cem\u3eIL1RL1\u3c/em\u3e genetic variant lowers soluble ST2 levels and the risk effects of \u3cem\u3eAPOE\u3c/em\u3e-ε4 in female patients with Alzheimer’s disease

Changes in the levels of circulating proteins are associated with Alzheimer’s disease (AD), whereas their pathogenic roles in AD are unclear. Here, we identified soluble ST2 (sST2), a decoy receptor of interleukin-33–ST2 signaling, as a new disease-causing factor in AD. Increased circulating sST2 level is associated with more severe pathological changes in female individuals with AD. Genome-wide association analysis and CRISPR–Cas9 genome editing identified rs1921622, a genetic variant in an enhancer element of IL1RL1, which downregulates gene and protein levels of sST2. Mendelian randomization analysis using genetic variants, including rs1921622, demonstrated that decreased sST2 levels lower AD risk and related endophenotypes in females carrying the Apolipoprotein E (APOE)-ε4 genotype; the association is stronger in Chinese than in European-descent populations. Human and mouse transcriptome and immunohistochemical studies showed that rs1921622/sST2 regulates amyloid-beta (Aβ) pathology through the modulation of microglial activation and Aβ clearance. These findings demonstrate how sST2 level is modulated by a genetic variation and plays a disease-causing role in females with AD

An IL1RL1 genetic variant lowers soluble ST2 levels and the risk effects of APOE-ε4 in female patients with Alzheimer’s disease

Changes in the levels of circulating proteins are associated with Alzheimer’s disease (AD), whereas their pathogenic roles in AD are unclear. Here, we identified soluble ST2 (sST2), a decoy receptor of interleukin-33–ST2 signaling, as a new disease-causing factor in AD. Increased circulating sST2 level is associated with more severe pathological changes in female individuals with AD. Genome-wide association analysis and CRISPR–Cas9 genome editing identified rs1921622, a genetic variant in an enhancer element of IL1RL1, which downregulates gene and protein levels of sST2. Mendelian randomization analysis using genetic variants, including rs1921622, demonstrated that decreased sST2 levels lower AD risk and related endophenotypes in females carrying the Apolipoprotein E (APOE)-ε4 genotype; the association is stronger in Chinese than in European-descent populations. Human and mouse transcriptome and immunohistochemical studies showed that rs1921622/sST2 regulates amyloid-beta (Aβ) pathology through the modulation of microglial activation and Aβ clearance. These findings demonstrate how sST2 level is modulated by a genetic variation and plays a disease-causing role in females with AD

An IL1RL1 genetic variant lowers soluble ST2 levels and the risk effects of APOE-ε4 in female patients with Alzheimer’s disease

Changes in the levels of circulating proteins are associated with Alzheimer’s disease (AD), whereas their pathogenic roles in AD are unclear. Here, we identified soluble ST2 (sST2), a decoy receptor of interleukin-33–ST2 signaling, as a new disease-causing factor in AD. Increased circulating sST2 level is associated with more severe pathological changes in female individuals with AD. Genome-wide association analysis and CRISPR–Cas9 genome editing identified rs1921622, a genetic variant in an enhancer element of IL1RL1, which downregulates gene and protein levels of sST2. Mendelian randomization analysis using genetic variants, including rs1921622, demonstrated that decreased sST2 levels lower AD risk and related endophenotypes in females carrying the Apolipoprotein E (APOE)-ε4 genotype; the association is stronger in Chinese than in European-descent populations. Human and mouse transcriptome and immunohistochemical studies showed that rs1921622/sST2 regulates amyloid-beta (Aβ) pathology through the modulation of microglial activation and Aβ clearance. These findings demonstrate how sST2 level is modulated by a genetic variation and plays a disease-causing role in females with AD

Administration of chitosan-tripolyphosphate-DNA nanoparticles overexpressing key enzymes to improve omega-3 long-chain polyunsaturated fatty acid synthesis in gilthead sea bream (Sparus aurata)

[eng] Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) known to prevent atherosclerosis, stroke, obesity, type-2 diabetes, inflammation and autoimmune disease, among others. Few organisms, such as Caenorhabditis elegans and some invertebrates, can synthesize the n-3 fatty acid series in significant amounts, and marine fish and shellfish, which acquire pre-formed LC-PUFA by trophic transfer, are the major sources of n-3 LC-PUFA in the human diet. However, substitution of fish oil by vegetable oils in aquafeeds reduces healthy n-3 LC-PUFA in cultured fish, while increases proinflammatory n-6 fatty acids. The present study aimed to empower Sparus aurata to boost endogenous synthesis of n-3 LC-PUFA by transient expression of fish codon-optimized Caenorhabditis elegans Δ12/n-6 (FAT-2) and Δ15/n-3 (FAT-1) fatty acid desaturases using chitosan-tripolyphosphate (TPP) nanoparticles as DNA delivery system. Growth performance, body composition, serum metabolites, fatty acid profile and expression of key enzymes in intermediary metabolism were evaluated in Sparus aurata juveniles 72 hours after a single intraperitoneal injection of chitosan-TPP nanoparticles encapsulated with expression plasmids encoding fish codon-optimized C. elegans FAT-1 and FAT-2 (short-term effect) and in 70-day treated fish that were periodically administered with chitosan-TPP-DNA nanoparticles (long- term sustained effect). For the short-term study, increased levels of FAT-1 and FAT-2 mRNAs (> 10-fold) were detected 72 hours post-injection in the S. aurata liver. Expression of FAT-1 elevated hepatic EPA and total n-3 PUFA. In addition to reduced serum triglycerides, co-expression of FAT-1 and FAT-2 also induced proportions of DHA and PUFA in the S. aurata liver. Compared to control fish, treatment with FAT-1, FAT-2 and FAT-1 + FAT-2 downregulated expression of hepatic key genes in glycolysis and lipogenesis, including g6pd, pfk1, pk, pfkfb1, acaca, acacb, fasn, scd1a and fads2. In addition, co-expression of FAT-1 and FAT-2 significantly upregulated the activity of rate-limiting enzymes in glycolysis and the pentose phosphate pathway. Fish expressing FAT-2 and FAT-1 + FAT-2 showed lower hepatic expression of pparg and srebf1, and higher of hnf4a. Treatment with FAT-1 and FAT-2 alone downregulated srebf1 and upregulated ppara, respectively, in the S. aurata liver. To study long-term sustained expression of FAT-1, FAT-2 and FAT-1 + FAT-2, chitosan-TPP-DNA nanoparticles were provided to fish every 4 weeks (3 doses in total). After 70 days of treatment, tissue distribution analysis showed high expression levels for FAT-1 and FAT-2 in the liver (>200-fold) followed by the intestine (10 to 25-fold), while no differential expression occurred in the skeletal muscle and brain. Expression of FAT-1 and FAT-1 + FAT-2 increased weight gain. Fatty acid methyl esters assay revealed that co-expression of FAT-1 and FAT-2 increased liver production and muscle accumulation of EPA, DHA and total n-3 LC-PUFA, while decreased the n-6/n-3 ratio. Co-expression of FAT-1 and FAT-2 downregulated srebf1 and genes encoding rate-limiting enzymes for de novo lipogenesis in the liver, leading to decreased circulating triglycerides and cholesterol. In contrast, FAT-2 and FAT-1 + FAT-2 upregulated hepatic hnf4a, nr1h3 and key enzymes in glycolysis and the pentose phosphate pathway. Our findings demonstrate that administration of chitosan-TPP-DNA nanoparticles, a methodology that circumvents obtention of genetically modified organisms, is a proper gene delivery method for sustained expression of exogenous enzymes in the liver of Sparus aurata. Specifically, co-expression of FAT-1 and FAT-2 enabled the production of functional fish for human consumption, rich in n-3 LC-PUFA, notably EPA and DHA, and with decreased n-6/n-3 fatty acids ratio. In addition, co- expression of FAT-1 and FAT-2 increased weight gain and the specific growth rate in Sparus aurata.[spa] Los ácidos eicosapentaenoico (20:5n-3, EPA) y docosahexaenoico (22:6n-3, DHA) son ácidos grasos poliinsaturados de cadena larga omega-3 (n-3 LC-PUFA) conocidos por prevenir aterosclerosis, accidentes cerebrovasculares, obesidad, diabetes tipo 2, inflamación y enfermedades autoinmunes, entre otras. Algunos organismos, como Caenorhabditis elegans y ciertos invertebrados, pueden sintetizar n-3 LC-PUFA en cantidades significativas. Peces y mariscos marinos adquieren LC-PUFA preformados por transferencia trófica y son las principales fuentes de n-3 LC-PUFA en la dieta humana. Sin embargo, la sustitución de aceite de pescado por aceites vegetales en piensos acuícolas reduce el contenido de n-3 LC-PUFA saludables en peces cultivados, mientras que aumenta la proporción de ácidos grasos n-6 proinflamatorios. El objetivo de esta tesis ha sido promover la biosíntesis de n-3 LC-PUFA en dorada (Sparus aurata) mediante la administración de nanopartículas de quitosano-tripolifosfato (TPP)-DNA para expresar transitoriamente las desaturasas de ácidos grasos Δ12/n-6 (FAT-2) y Δ15/n-3 (FAT- 1) de Caenorhabditis elegans. Se evaluó el efecto sobre el crecimiento, composición corporal, metabolitos séricos, perfil de ácidos grasos y expresión de enzimas clave en el metabolismo intermediario en juveniles de S. aurata 72 horas después de una dosis intraperitoneal de nanopartículas de quitosano-TPP encapsuladas con plásmidos de expresión de FAT-1 y FAT-2 de C. elegans con codones optimizados para peces (efecto a corto plazo) y en peces administrados periódicamente con nanopartículas de quitosano-TPP-DNA durante 70 días (efecto sostenido a largo plazo). A corto plazo, 72 horas tras la administración de las nanopartículas, los niveles de mRNA de FAT-1 y FAT-2 se incrementaron en hígado de S. aurata (> 10 veces). La expresión de FAT-1 incrementó EPA y n-3 PUFA total en hígado. La coexpresión de FAT-1 y FAT-2 redujo los triglicéridos séricos e incrementó las proporciones de DHA y PUFA hepáticas. En comparación con peces control, el tratamiento con FAT-1, FAT-2 y FAT-1 + FAT-2 disminuyó la expresión de genes hepáticos clave en glucólisis y lipogénesis, como g6pd, pfkl, pklr, pfkfb1, acaca, acacb, fasn, scd1a y fads2. Además, la coexpresión de FAT-1 y FAT-2 aumentó significativamente la actividad de enzimas clave en glucólisis y vía de las pentosas fosfato. FAT-2 y FAT-1 + FAT-2 disminuyeron la expresión hepática de pparg y srebf1, e incrementaron hnf4a. El tratamiento con FAT-1 disminuyó srebf1 mientras que FAT-2 incrementó ppara en hígado de S. aurata. La expresión sostenida de FAT-1, FAT-2 y FAT-1 + FAT-2 a largo plazo se estudió en peces tratados con nanopartículas de quitosano-TPP-DNA cada 4 semanas (3 dosis en total). Tras 70 días, el análisis de la distribución tisular mostró niveles elevados de expresión de FAT-1 y FAT-2 en hígado (>200 veces) e intestino (10-25 veces), no hallándose expresión diferencial en músculo esqueleto ni cerebro. La expresión de FAT-1 y FAT-1 + FAT-2 incrementó la tasa de crecimiento. El ensayo de ésteres metílicos de ácidos grasos reveló que la coexpresión de FAT-1 y FAT-2 incrementó la producción hepática y acumulación muscular de EPA, DHA y n-3 LC-PUFA total, disminuyendo la relación n-6/n-3. La coexpresión de FAT-1 y FAT-2 disminuyó la expresión de srebf1 y enzimas clave en lipogénesis de novo hepática, causando una disminución de triglicéridos y colesterol circulantes. Por el contrario, FAT-2 y FAT-1 + FAT-2 incrementaron la expresión de hnf4a, nr1h3 y enzimas clave en glucólisis y ruta de las pentosas fosfato. Nuestros resultados muestran que las nanopartículas de quitosano-TPP-DNA promueven la expresión sostenida de enzimas exógenas en hígado de S. aurata, evitando la generación de organismos modificados genéticamente. Específicamente, la coexpresión de FAT-1 y FAT-2 permite producir pescado funcional para consumo rico en n-3 LC-PUFA, particularmente EPA y DHA, y con baja relación n-6/n-3. Además, la coexpresión de FAT-1 y FAT-2 incrementó la talla y la tasa de crecimiento específico en S. aurata

Administration of chitosan-tripolyphosphate-DNA nanoparticles overexpressing key enzymes to improve omega-3 long-chain polyunsaturated fatty acid synthesis in gilthead sea bream (Sparus aurata)

Programa de Doctorat en Biotecnologia[eng] Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) known to prevent atherosclerosis, stroke, obesity, type-2 diabetes, inflammation and autoimmune disease, among others. Few organisms, such as Caenorhabditis elegans and some invertebrates, can synthesize the n-3 fatty acid series in significant amounts, and marine fish and shellfish, which acquire pre-formed LC-PUFA by trophic transfer, are the major sources of n-3 LC-PUFA in the human diet. However, substitution of fish oil by vegetable oils in aquafeeds reduces healthy n-3 LC-PUFA in cultured fish, while increases proinflammatory n-6 fatty acids. The present study aimed to empower Sparus aurata to boost endogenous synthesis of n-3 LC-PUFA by transient expression of fish codon-optimized Caenorhabditis elegans Δ12/n-6 (FAT-2) and Δ15/n-3 (FAT-1) fatty acid desaturases using chitosan-tripolyphosphate (TPP) nanoparticles as DNA delivery system. Growth performance, body composition, serum metabolites, fatty acid profile and expression of key enzymes in intermediary metabolism were evaluated in Sparus aurata juveniles 72 hours after a single intraperitoneal injection of chitosan-TPP nanoparticles encapsulated with expression plasmids encoding fish codon-optimized C. elegans FAT-1 and FAT-2 (short-term effect) and in 70-day treated fish that were periodically administered with chitosan-TPP-DNA nanoparticles (long- term sustained effect). For the short-term study, increased levels of FAT-1 and FAT-2 mRNAs (> 10-fold) were detected 72 hours post-injection in the S. aurata liver. Expression of FAT-1 elevated hepatic EPA and total n-3 PUFA. In addition to reduced serum triglycerides, co-expression of FAT-1 and FAT-2 also induced proportions of DHA and PUFA in the S. aurata liver. Compared to control fish, treatment with FAT-1, FAT-2 and FAT-1 + FAT-2 downregulated expression of hepatic key genes in glycolysis and lipogenesis, including g6pd, pfk1, pk, pfkfb1, acaca, acacb, fasn, scd1a and fads2. In addition, co-expression of FAT-1 and FAT-2 significantly upregulated the activity of rate-limiting enzymes in glycolysis and the pentose phosphate pathway. Fish expressing FAT-2 and FAT-1 + FAT-2 showed lower hepatic expression of pparg and srebf1, and higher of hnf4a. Treatment with FAT-1 and FAT-2 alone downregulated srebf1 and upregulated ppara, respectively, in the S. aurata liver. To study long-term sustained expression of FAT-1, FAT-2 and FAT-1 + FAT-2, chitosan-TPP-DNA nanoparticles were provided to fish every 4 weeks (3 doses in total). After 70 days of treatment, tissue distribution analysis showed high expression levels for FAT-1 and FAT-2 in the liver (>200-fold) followed by the intestine (10 to 25-fold), while no differential expression occurred in the skeletal muscle and brain. Expression of FAT-1 and FAT-1 + FAT-2 increased weight gain. Fatty acid methyl esters assay revealed that co-expression of FAT-1 and FAT-2 increased liver production and muscle accumulation of EPA, DHA and total n-3 LC-PUFA, while decreased the n-6/n-3 ratio. Co-expression of FAT-1 and FAT-2 downregulated srebf1 and genes encoding rate-limiting enzymes for de novo lipogenesis in the liver, leading to decreased circulating triglycerides and cholesterol. In contrast, FAT-2 and FAT-1 + FAT-2 upregulated hepatic hnf4a, nr1h3 and key enzymes in glycolysis and the pentose phosphate pathway. Our findings demonstrate that administration of chitosan-TPP-DNA nanoparticles, a methodology that circumvents obtention of genetically modified organisms, is a proper gene delivery method for sustained expression of exogenous enzymes in the liver of Sparus aurata. Specifically, co-expression of FAT-1 and FAT-2 enabled the production of functional fish for human consumption, rich in n-3 LC-PUFA, notably EPA and DHA, and with decreased n-6/n-3 fatty acids ratio. In addition, co- expression of FAT-1 and FAT-2 increased weight gain and the specific growth rate in Sparus aurata.[spa] Los ácidos eicosapentaenoico (20:5n-3, EPA) y docosahexaenoico (22:6n-3, DHA) son ácidos grasos poliinsaturados de cadena larga omega-3 (n-3 LC-PUFA) conocidos por prevenir aterosclerosis, accidentes cerebrovasculares, obesidad, diabetes tipo 2, inflamación y enfermedades autoinmunes, entre otras. Algunos organismos, como Caenorhabditis elegans y ciertos invertebrados, pueden sintetizar n-3 LC-PUFA en cantidades significativas. Peces y mariscos marinos adquieren LC-PUFA preformados por transferencia trófica y son las principales fuentes de n-3 LC-PUFA en la dieta humana. Sin embargo, la sustitución de aceite de pescado por aceites vegetales en piensos acuícolas reduce el contenido de n-3 LC-PUFA saludables en peces cultivados, mientras que aumenta la proporción de ácidos grasos n-6 proinflamatorios. El objetivo de esta tesis ha sido promover la biosíntesis de n-3 LC-PUFA en dorada (Sparus aurata) mediante la administración de nanopartículas de quitosano-tripolifosfato (TPP)-DNA para expresar transitoriamente las desaturasas de ácidos grasos Δ12/n-6 (FAT-2) y Δ15/n-3 (FAT- 1) de Caenorhabditis elegans. Se evaluó el efecto sobre el crecimiento, composición corporal, metabolitos séricos, perfil de ácidos grasos y expresión de enzimas clave en el metabolismo intermediario en juveniles de S. aurata 72 horas después de una dosis intraperitoneal de nanopartículas de quitosano-TPP encapsuladas con plásmidos de expresión de FAT-1 y FAT-2 de C. elegans con codones optimizados para peces (efecto a corto plazo) y en peces administrados periódicamente con nanopartículas de quitosano-TPP-DNA durante 70 días (efecto sostenido a largo plazo). A corto plazo, 72 horas tras la administración de las nanopartículas, los niveles de mRNA de FAT-1 y FAT-2 se incrementaron en hígado de S. aurata (> 10 veces). La expresión de FAT-1 incrementó EPA y n-3 PUFA total en hígado. La coexpresión de FAT-1 y FAT-2 redujo los triglicéridos séricos e incrementó las proporciones de DHA y PUFA hepáticas. En comparación con peces control, el tratamiento con FAT-1, FAT-2 y FAT-1 + FAT-2 disminuyó la expresión de genes hepáticos clave en glucólisis y lipogénesis, como g6pd, pfkl, pklr, pfkfb1, acaca, acacb, fasn, scd1a y fads2. Además, la coexpresión de FAT-1 y FAT-2 aumentó significativamente la actividad de enzimas clave en glucólisis y vía de las pentosas fosfato. FAT-2 y FAT-1 + FAT-2 disminuyeron la expresión hepática de pparg y srebf1, e incrementaron hnf4a. El tratamiento con FAT-1 disminuyó srebf1 mientras que FAT-2 incrementó ppara en hígado de S. aurata. La expresión sostenida de FAT-1, FAT-2 y FAT-1 + FAT-2 a largo plazo se estudió en peces tratados con nanopartículas de quitosano-TPP-DNA cada 4 semanas (3 dosis en total). Tras 70 días, el análisis de la distribución tisular mostró niveles elevados de expresión de FAT-1 y FAT-2 en hígado (>200 veces) e intestino (10-25 veces), no hallándose expresión diferencial en músculo esqueleto ni cerebro. La expresión de FAT-1 y FAT-1 + FAT-2 incrementó la tasa de crecimiento. El ensayo de ésteres metílicos de ácidos grasos reveló que la coexpresión de FAT-1 y FAT-2 incrementó la producción hepática y acumulación muscular de EPA, DHA y n-3 LC-PUFA total, disminuyendo la relación n-6/n-3. La coexpresión de FAT-1 y FAT-2 disminuyó la expresión de srebf1 y enzimas clave en lipogénesis de novo hepática, causando una disminución de triglicéridos y colesterol circulantes. Por el contrario, FAT-2 y FAT-1 + FAT-2 incrementaron la expresión de hnf4a, nr1h3 y enzimas clave en glucólisis y ruta de las pentosas fosfato. Nuestros resultados muestran que las nanopartículas de quitosano-TPP-DNA promueven la expresión sostenida de enzimas exógenas en hígado de S. aurata, evitando la generación de organismos modificados genéticamente. Específicamente, la coexpresión de FAT-1 y FAT-2 permite producir pescado funcional para consumo rico en n-3 LC-PUFA, particularmente EPA y DHA, y con baja relación n-6/n-3. Además, la coexpresión de FAT-1 y FAT-2 incrementó la talla y la tasa de crecimiento específico en S. aurata