Intracellular delivery of nucleic acids using a new type of cell penetrating peptides

[ES]La terapia génica ha cautivado a la comunidad científica debido a su potencial para tratar las enfermedades genéticas: si un gen se expresa en exceso se inactiva, y si se expresa poco o de forma incorrecta se reemplaza por un gen funcional. Asimismo, también se puede emplear para tratar enfermedades tan prevalentes como el cáncer, lo que incrementa su interés. Su fundamento parece muy sencillo, pero la realidad es que el diseño de vehículos transportadores que permitan hacer llegar esta terapia al lugar de acción supone un gran desafío: es necesario superar todas las barreras fisiológicas de forma eficiente, sin causar toxicidad, y preferentemente de forma selectiva sobre el tejido diana. En este trabajo se presentan nuevos vectores basados en péptidos penetrantes de membrana para la entrega intracelular de plásmidos y RNA interferente. Estos péptidos tienen una carga neta positiva, presentan una estructura anfipática secundaria, y han sido modificados químicamente mediante el acoplamiento de aldehídos de cadena larga a través de un enlace hidrazona. Se realizó su síntesis y caracterización, y se realizaron ensayos de transfección y de citotoxicidad in vitro. Además, se analizó la influencia que ejercía la naturaleza del cargo y la posición de los aldehídos del péptido penetrante sobre la eficiencia de transfección[EN]Gene therapy has attracted the interest of the scientific community, due to its potential to treat genetic diseases: it works by inactivating overexpressed genes or by replacing malfunctioning ones. In addition, this therapy can be used to cure other diseases such as cancer. However, its strong potential is hindered by the delivery problem: designing vehicles that allow the efficient transportation of therapeutic nucleic acids to its target tissue is a considerable challenge, since many physiological barriers must be overcome in an efficient way, and without causing cytotoxicity. A new type of cell penetrating peptide-based vectors for the intracellular delivery of plasmids and short interfering RNAs (siRNAs) is presented in this work. These vectors have an amphiphilic secondary structure, are positively charged, and are chemically modified by coupling long chain aliphatic aldehydes through a hydrazone bond. They have been synthesized and characterized in the laboratory; and in vitro assays have been performed in order to study their transfection efficiency and cytotoxicity. Furthermore, the influence of different factors on transfection efficiency has been studied, such as the position of the aldehyde pendants and the nature of the nucleotide carg

Dendritic Cell‐Mediated Cross‐Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS‐CoV‐2

SARS-CoV-2 B.1.351 and B.1.167.2 viruses used in this study were obtained through the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 653316. SARS-CoV-2 B.1 (MAD6 isolate) was kindly provided by José M. Honrubia and Luis Enjuanes (CNB-CSIC, Madrid, Spain). The authors thank Centro de Investigación en Sanidad Animal (CISA)-Instituto Nacional de Investigaciones Agrarias (INIA-CSIC) (Valdeolmos, Madrid, Spain) for the BSL-3 facilities. Research in LAV laboratory was funded by the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 y COVID19); the MCIN/AEI/10.13039/501100011033 (PID2020-117323RB-I00 and PDC2021-121711-I00), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (DTS20/00089), partially supported by the ERDF, the Spanish Association Against Cancer (AECC 19084); the CRIS Cancer Foundation (FCRISIFI-2018 and FCRIS-2021-0090), the Fundación Caixa-Health Research (HR21-00761 project IL7R_LungCan), and the Comunidad de Madrid (P2022/BMD-7225 NEXT_GEN_CART_MAD-CM). Work in the DS laboratory was funded by the CNIC; the European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091; MCIN/AEI/10.13039/501100011033 (PID2019-108157RB); Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); Atresmedia (Constantes y Vitales prize); Fondo Solidario Juntos (Banco Santander); and “La Caixa” Foundation (LCF/PR/HR20/00075). The CNIC was supported by the ISCIII, the MCIN and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020- 001041-S funded by MCIN/AEI/10.13039/501100011033). Research in RD laboratory was supported by the ISCIII (PI2100989) and CIBERINFEC; the European Commission Horizon 2020 Framework Programme (grant numbers 731868 project VIRUSCAN FETPROACT-2016, and 101046084 project EPIC-CROWN-2); and the Fundación CaixaHealth Research (grant number HR18-00469 project StopEbola). Research in CNB-CSIC laboratory was funded by Fondo Supera COVID19 (Crue Universidades-Banco Santander) grant, CIBERINFEC, and Spanish Research Council (CSIC) grant 202120E079 (to J.G.-A.), CSIC grant 2020E84 (to M.E.), MCIN/AEI/10.13039/501100011033 (PID2020- 114481RB-I00 to J.G-A. and M.E.), and by the European CommissionNextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global) to J.G.-A. and M.E. Work in the CIB-CSIC laboratory was supported by MCIN/AEI/10.13039/501100011033 (PID2019-104544GB-I00 and 2023AEP105 to CA, and PID2020-113225GB-I00 to F.J.B.). Cryo-EM data were collected at the Maryland Center for Advanced Molecular Analyses which was supported by MPOWER (The University of Maryland Strategic Partnership). I.H.-M. receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 71367. L.R.-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck.S

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2

17 p.-4 fig.Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT, are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT, the bispecific trimerbody TNTDNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNTDNGR-1, but not TNT, protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.SARS-CoV-2 B.1.351 and B.1.167.2 viruses used in this study were obtained through the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 653316. SARS-CoV-2 B.1 (MAD6 isolate) was kindly provided by José M. Honrubia and Luis Enjuanes (CNB-CSIC, Madrid, Spain). The authors thank Centro de Investigación en Sanidad Animal (CISA)-Instituto Nacional de Investigaciones Agrarias (INIA-CSIC) (Valdeolmos, Madrid, Spain) for the BSL-3 facilities. Research in LA-V laboratory was funded by the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 y COVID-19); the MCIN/AEI/10.13039/501100011033 (PID2020-117323RB-I00 and PDC2021-121711-I00), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (DTS20/00089), partially supported by the ERDF, the Spanish Association Against Cancer (AECC 19084); the CRIS Cancer Foundation (FCRIS-IFI-2018 and FCRIS-2021-0090), the Fundación Caixa-Health Research (HR21-00761 project IL7R_LungCan), and the Comunidad de Madrid (P2022/BMD-7225 NEXT_GEN_CART_MAD-CM). Work in the DS laboratory was funded by the CNIC; the European Union's Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091; MCIN/AEI/10.13039/501100011033 (PID2019-108157RB); Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); Atresmedia (Constantes y Vitales prize); Fondo Solidario Juntos (Banco Santander); and “La Caixa” Foundation (LCF/PR/HR20/00075). The CNIC was supported by the ISCIII, the MCIN and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011033). Research in RD laboratory was supported by the ISCIII (PI2100989) and CIBERINFEC; the European Commission Horizon 2020 Framework Programme (grant numbers 731868 project VIRUSCAN FETPROACT-2016, and 101046084 project EPIC-CROWN-2); and the Fundación Caixa-Health Research (grant number HR18-00469 project StopEbola). Research in CNB-CSIC laboratory was funded by Fondo Supera COVID-19 (Crue Universidades-Banco Santander) grant, CIBERINFEC, and Spanish Research Council (CSIC) grant 202120E079 (to J.G.-A.), CSIC grant 2020E84 (to M.E.), MCIN/AEI/10.13039/501100011033 (PID2020-114481RB-I00 to J.G-A. and M.E.), and by the European Commission-NextGenerationEU, through CSIC's Global Health Platform (PTI Salud Global) to J.G.-A. and M.E. Work in the CIB-CSIC laboratory was supported by MCIN/AEI/10.13039/501100011033 (PID2019-104544GB-I00 and 2023AEP105 to CA, and PID2020-113225GB-I00 to F.J.B.). Cryo-EM data were collected at the Maryland Center for Advanced Molecular Analyses which was supported by MPOWER (The University of Maryland Strategic Partnership). I.H.-M. receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 71367. L.R.-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck.Peer reviewe