Nuevas estrategias de compactación y transfección de DN-RNA en terapia génica

La Terapia Génica pretende tratar enfermedades hereditarias o adquiridas, a nivel molecular, bien reparando posibles daños del DNA celular, mediante la inserción de plásmidos de DNA (pDNA) en el citoplasma de las células dañadas, o bloqueando (silenciando) la función patogénica de algún gen mediante la inserción de RNAs pequeños de interferencia (small interferring RNA o siRNA). Entre los distintos métodos para transfectar ácidos nucleicos al interior celular, los sistemas autoagregados coloidales y/o supramoleculares se han revelado en los últimos tiempos como una alternativa plausible a los vectores víricos, habitualmente utilizados en el pasado. En particular, los lípidos catiónicos (CLs) o aniónicos (ALs), las NPs inorgánicas magnéticas o no magnéticas, ciclos supramoleculares (calixarenos CXs o pilararenos PLs) o las ciclodextrinas polianfifílicas (paCDs) son capaces de compactar DNA mediante una interacción electrostática superficial entrópicamente dirigida, y transfectarlo al interior de las células dañadas. Sin embargo, a pesar de todo lo que ya se conoce de este proceso, quedan todavía ciertos problemas por resolver, como los bajos niveles de transfección celular y la relativamente alta citotoxicidad de estos vectores en entornos celulares. Además, se sabe poco acerca del proceso de liberación del ácido nucleico una vez el vector ha traspasado la membrana plasmática, por lo que se tiene escaso o nulo control sobre el potencial de un determinado sistema vehiculizador como agente de transfección in vitro y, finalmente, in vivo. Es por ello que se está dedicando actualmente una especial atención dentro de este campo de investigación a intentar entender cómo se libera el agente terapéutico en el interior de la célula y cuáles son las barreras fisiológicas que puede encontrarse en su camino hacia el núcleo. Sin duda, conseguir el objetivo de poder curar enfermedades con nuevos protocolos de terapia génica pasa por tener un profundo conocimiento y control sobre ambas etapas del proceso de transfección: la primera, centrada en una buena compactación y transporte del ácido nucleico a través de la membrana, y la segunda, dirigida a la liberación del material genético en el entorno celular adecuado..

Multidisciplinary approach to the transfection of plasmid DNA by a nonviral nanocarrier based on a Gemini-Bolaamphiphilic hybrid lipid

A multidisciplinary strategy, including bothbiochemical and biophysical studies, was proposed here toevaluate the potential of lipid nanoaggregates consisting of amixture of a gemini−bolaamphiphilic lipid (C6C22C6) and thewell-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidy-lethanolamine (DOPE) to transfect plasmid DNA into livingcells in an efficient and safe way. For that purpose, severalexperimental techniques were employed, such as zeta potential(phase analysis light scattering methodology), agarose gelelectrophoresis (pDNA compaction and pDNA protectionassays), small-angle X-ray scattering, cryo-transmission electronmicroscopy, atomic force microscopy,fluorescence-assisted cellsorting, luminometry, and cytotoxicity assays. The resultsrevealed that the cationic lipid and plasmid offer only 70 and30% of their nominal positive (=++q2.0nom,C C C6226) and negative charges (=−−q2/bpnom,pDNA), respectively. Upon mixing withDOPE, they form lipoplexes that self-aggregate in typical multilamellar Lαlyotropic liquid-crystal nanostructures with sizes in therange of 100−200 nm and low polydispersities, very suitablyfitted to remain in the bloodstream and cross the cell membrane.Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNAplasmids (pEGFP-C3, encoding the greenfluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with anefficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/−charge ratio wasmaintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic becausethe viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosysteman optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experimentsFinancial support from the Ministerio de Economia y Competitividad of Spain (projects CTQ2012-30821, CTQ2015-65972-R, CTQ2015-64425-C2-2-R, and CTQ2014-55208-P), Madrid Regional Government (S2013/MIT-2807), Xunta de Galicia (GR 2007/085; IN607C 2016/03 and Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09), the European Regional Development Fund (ERDF), and Universidad Complutense de Madrid, Spain (project UCMA05-33-010), is gratefully acknowledgedS

Intercellular Trafficking of Gold Nanostars in Uveal Melanoma Cells for Plasmonic Photothermal Therapy

Efficient plasmonic photothermal therapies (PPTTs) using non-harmful pulse laser irradiation at the near-infrared (NIR) are a highly sought goal in nanomedicine. These therapies rely on the use of plasmonic nanostructures to kill cancer cells while minimizing the applied laser power density. Cancer cells have an unsettled capacity to uptake, retain, release, and re-uptake gold nanoparticles, thus offering enormous versatility for research. In this work, we have studied such cell capabilities for nanoparticle trafficking and its impact on the effect of photothermal treatments. As our model system, we chose uveal (eye) melanoma cells, since laser-assisted eye surgery is routinely used to treat glaucoma and cataracts, or vision correction in refractive surgery. As nanostructure, we selected gold nanostars (Au NSs) due to their high photothermal efficiency at the near-infrared (NIR) region of the electromagnetic spectrum. We first investigated the photothermal effect on the basis of the dilution of Au NSs induced by cell division. Using this approach, we obtained high PPTT efficiency after several cell division cycles at an initial low Au NS concentration (pM regime). Subsequently, we evaluated the photothermal effect on account of cell division upon mixing Au NS-loaded and non-loaded cells. Upon such mixing, we observed trafficking of Au NSs between loaded and non-loaded cells, thus achieving effective PPTT after several division cycles under low irradiation conditions (below the maximum permissible exposure threshold of skin). Our study reveals the ability of uveal melanoma cells to release and re-uptake Au NSs that maintain their plasmonic photothermal properties throughout several cell division cycles and re-uptake. This approach may be readily extrapolated to real tissue and even to treat in situ the eye tumor itself. We believe that our method can potentially be used as co-therapy to disperse plasmonic gold nanostructures across affected tissues, thus increasing the effectiveness of classic PPTT

Protein Expression Knockdown in Cancer Cells Induced by a Gemini Cationic Lipid Nanovector with Histidine-Based Polar Heads

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in Lα lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presentedThis work has been funded by the Spanish Ministry of Science, Innovation and Universities (MICIU) (Grant RTI2018-095844-B-I00 and CTQ2017-88948-P), the University Complutense of Madrid (Spain) (project number UCMA05-33-010), and the Regional Government of Madrid (Grant P2018/NMT-4389). P.T. thanks Agencia Estatal de Investigación (AEI) through the Project MAT2016-80266-R and Xunta de Galicia (Grupo de Referencia Competitiva ED431C 2018/26; Agrupación Estratégica en Materiales-AEMAT ED431E 2018/08). ERDF funds are all greatly acknowledged. The proteomic analysis was performed in the Proteomics Unit of Complutense University of Madrid, a member of ProteoRed and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDFS

Multidisciplinary Approach to the Transfection of Plasmid DNA by a Nonviral Nanocarrier Based on a Gemini-Bolaamphiphilic Hybrid Lipid

A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini-bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive () and negative charges (), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100-200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/- charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*

Clinical Presentation, Management, and Evolution of Lymphomas in Patients with Inflammatory Bowel Disease: An ENEIDA Registry Study

Inflammatory bowel disease; Lymphoma; ThiopurinesEnfermedad inflamatoria intestinal; Linfoma; TiopurinasMalaltia inflamatòria de l'intestí; Limfoma; TiopurinesAn increased risk of lymphoma has been described in patients with inflammatory bowel disease (IBD). The aims of our study were to determine the clinical presentation, the previous exposure to immunosuppressive and biologic therapies, and the evolution of lymphomas in patients with IBD. IBD patients with diagnosis of lymphoma from October 2006 to June 2021 were identified from the prospectively maintained ENEIDA registry of GETECCU. We identified 52 patients (2.4 cases of lymphoma/1000 patients with IBD; 95% CI 1.8-3.1). Thirty-five were men (67%), 52% had ulcerative colitis, 60% received thiopurines, and 38% an anti-TNF drug before lymphoma diagnosis. Age at lymphoma was lower in those patients treated with thiopurines (53 ± 17 years old) and anti-TNF drugs (47 ± 17) than in those patients not treated with these drugs (63 ± 12; p < 0.05). Five cases had relapse of lymphoma (1.7 cases/100 patient-years). Nine patients (17%) died after 19 months (IQR 0-48 months). Relapse and mortality were not related with the type of IBD or lymphoma, nor with thiopurines or biologic therapies. In conclusion, most IBD patients had been treated with thiopurines and/or anti-TNF agents before lymphoma diagnosis, and these patients were younger at diagnosis of lymphoma than those not treated with these drugs. Relapse and mortality of lymphoma were not related with these therapies

Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12

Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors for IL-12 delivery. This study was conceived to further endorse the use of gemini cationic lipids (GCLs) in combination with zwitterionic helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine) as nanovectors for the insertion of plasmid DNA encoding for IL-12 (pCMV-IL12) into cells. Optimal GCL formulations previously reported by us were selected for IL-12-based biophysical experiments. In vitro studies demonstrated efficient pCMV-IL12 transfection by GCLs with comparable or superior cytokine levels than those obtained with commercial control Lipofectamine2000*. Furthermore, the nanovectors did not present significant toxicity, showing high cell viability values. The proteins adsorbed on the nanovector surface were found to be mostly lipoproteins and serum albumin, which are both beneficial to increase the blood circulation time. These outstanding results are accompanied by an initial physicochemical characterization to confirm DNA compaction and protection by the lipid mixture. Although further studies would be necessary, the present GCLs exhibit promising characteristics as candidates for pCMV-IL12 transfection in future in vivo applications

A gemini cationic lipid with histidine residues as a novel lipid-based gene nanocarrier: a biophysical and biochemical study

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120–290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies

Characterizing the phenotype and mode of inheritance of patients with inherited peripheral neuropathies carrying MME mutations

[EN] Background Mutations in the metalloendopeptidase (MME) gene were initially identified as a cause of autosomal recessive Charcot-Marie-Tooth disease type 2 (CMT2). Subsequently, variants in MME were linked to other late-onset autosomal dominant polyneuropathies. Thus, our goal was to define the phenotype and mode of inheritance of patients carrying changes in MME. Methods We screened 197 index cases with a hereditary neuropathy of the CMT type or distal hereditary motor neuropathy (dHMN) and 10 probands with familial amyotrophic lateral sclerosis (fALS) using a custom panel of 119 genes. In addition to the index case subjects, we also studied other clinically and/or genetically affected and unaffected family members. Results We found 17 variants in MME in a total of 20 index cases, with biallelic MME mutations detected in 13 cases from nine families (three in homozygosis and six in compound heterozygosis) and heterozygous variants found in 11 families. All patients with biallelic variants had a similar phenotype, consistent with late-onset axonal neuropathy. Conversely, the phenotype of patients carrying heterozygous mutations was highly variable [CMT type 1 (CMT1), CMT2, dHMN and fALS] and mutations did not segregate with the disease. Conclusion MME mutations that segregate in an autosomal recessive pattern are associated with a late-onset CMT2 phenotype, yet we could not demonstrate that MME variants in heterozygosis cause neuropathy. Our data highlight the importance of establishing an accurate genetic diagnosis in patients carrying MME mutations, especially with a view to genetic counselling.The authors thank the patients and healthy relatives for having participated in this project. We are grateful to the Eurobiobank CIBERER and the Biobank La Fe for their participation in the collection and processing of patient samples. We also thank the technicians at the Department of Genomics and Translational Genetics (CIPF) who participated in the quality control and processing of DNA samples (Virginia Rejas and Laura Ramírez), and the Bachelor¿s thesis student Andrea Ballester who helped with some clinical data collection. This project was funded by the Instituto de Salud Carlos III (ISCIII), FEDER (Grants no. PI12/00946 and PI16/00403 to TS, PI15/00187 to CE). MF holds a grant funded by the IIS La Fe (Grant no. 2015/0085). AS-M holds a grant funded by the Fundació Per Amor a l'Art (FPAA). JFV-C holds a ' Rio Hortega' contract funded by the ISCIII.Lupo, V.; Frasquet, M.; Sánchez-Monteagudo, A.; Pelayo-Negro, A.; García-Sobrino, T.; Sedano, MJ.; Pardo, J.... (2018). Characterizing the phenotype and mode of inheritance of patients with inherited peripheral neuropathies carrying MME mutations. Journal of Medical Genetics. 55(12):814-823. https://doi.org/10.1136/jmedgenet-2018-105650814823551