Nombre de maladies ont pour origine une absence d’expression ou une expression défectueuse d’une protéine donnée. Pour certaines d’entre elles, la protéine faisant défaut est circulante et peut être captée par les cellules lorsqu’elle est délivrée de façon exogène. Dans ce cas, les thérapies ont d’abord consisté en l’administration de la protéine thérapeutique extraite de tissus humains. Par la suite, le génie génétique a permis la production des protéines par fermentation de cellules après y avoir introduit le gène correspondant. Pour beaucoup d’autres maladies, la protéine faisant défaut ne peut être délivrée de façon exogène. Une production endogène de la protéine thérapeutique, par les cellules elles-mêmes est donc nécessaire. La technologie de l’ARN messager (ARNm), comme celle la précédant de l’ADN, se propose de supplémenter, au cœur des cellules, l’information génétique nécessaire pour produire elles-mêmes la protéine thérapeutique. Cependant, contrairement aux thérapies utilisant l’ADN, le transfert d’ARNm permet une expression transitoire de la protéine d’intérêt ce qui constitue un avantage dans nombre de maladies. La maîtrise de la quantité, de la qualité et de la régulation spatio-temporelle de la production d’une protéine codée par l’ARNm thérapeutique représente, néanmoins, un défi important pour le développement de cette approche.

Many diseases originate from either the absence or defective expression of a given protein. For some of them, the lacking protein is secreted or can be taken up by cells when delivered exogenously. In such cases, therapies initially involved administering the physiological protein extracted from human tissues. Subsequently, genetic engineering enabled the production of proteins through cell fermentation after introducing the corresponding gene. For many other pathologies, the deficient protein cannot be delivered exogenously. Thus, an endogenous production of the therapeutic protein by the cells themselves is necessary. Messenger RNA (mRNA) technology, like its predecessor DNA, aims to supplement the genetic information needed to produce the therapeutic protein within the cells. However, unlike DNA-based therapies, mRNA transfer allows for transient expression of the protein of interest, which offers an advantage in numerous pathologies. Nonetheless, mastering the quantity, quality, and spatio-temporal regulation of protein production encoded by therapeutic mRNA remains a significant challenge for the development of this approach.

Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses co-circulating in the same endemic areas. Infection can raise cross-reactive antibodies that can be either protective or increase risk of severe disease, depending on the infection sequence, DENV serotype and elapsed time between infection. On the contrast, T cell-mediated immunity against DENV and ZIKV is considered protective. Therefore, we have developed a T cell vaccine enriched in immunodominant T cell epitopes derived from ZIKV and evaluated its immunogenicity and efficacy against ZIKV and DENV infection. Mice were vaccinated using DNA vaccine platform using the tetrafunctional amphiphilic block copolymer 704. We show that vaccination of 2 different HLA class I transgenic mice with the ZIKV non-structural (NS) poly-epitope elicits T cell response against numerous ZIKV epitopes. Moreover, vaccination induces a significant protection against ZIKV infection, in the absence of neutralizing or enhancing antibodies against ZIKV. However, vaccination does not induce a significant protection against DENV2. In contrast, immunization with a DENV1-NS poly-epitope induces a significant protection against both DENV1 and DENV2, in the absence of humoral immunity. Taken together, we have shown that T-cell based vaccination could protect against multiple flavivirus infections and could overcome the complexity of antibody-mediated enhancement.

Genetic immunization is an attractive approach for prophylactic and therapeutic vaccination using synthetic vectors to deliver antigen-encoding nucleic acids. Recently, DNA delivered by a physical means or RNA by liposomes consisting of four different lipids demonstrated good protection in human phase III clinical trials and received Drugs Controller General of India and US FDA approval to protect against COVID-19, respectively. However, the development of a system allowing for efficient and simple delivery of nucleic acids while improving immune response priming has the potential to unleash the full therapeutic potential of genetic immunization. DNA-based gene therapies and vaccines have the potential for rapid development, as exemplified by the recent approval of Collategene, a gene therapy to treat human critical limb ischemia, and ZyCoV, a DNA vaccine delivered by spring-powered jet injector to protect against SARS-CoV2 infection. Recently, we reported amphiphilic block copolymer 704 as a promising synthetic vector for DNA vaccination in various models of human diseases. This vector allows dose sparing of antigen-encoding plasmid DNA. Here, we report the capacity of 704-mediated HIV and anti-hepatocellular carcinoma DNA vaccines to induce the production of specific antibodies against gp120 HIV envelope proteins in mice and against alpha-fetoprotein antigen in non-human primates, respectively. An investigation of the underlying mechanisms showed that 704-mediated vaccination did trigger a strong immune response by (1) allowing a direct DNA delivery into the cytosol, (2) promoting an intracytoplasmic DNA sensing leading to both interferon and NF-κB cascade stimulation, and (3) inducing antigen expression by muscle cells and presentation by antigen-presenting cells, leading to the induction of a robust adaptive response. Overall, our findings suggest that the 704-mediated DNA vaccination platform is an attractive method to develop both prophylactic and therapeutic vaccines.

Although tocilizumab treatment in severe and critical coronavirus disease 2019 (COVID-19) patients has proven its efficacy at the clinical level, there is little evidence supporting the effect of short-term use of interleukin-6 receptor blocking therapy on the B cell sub-populations and the cross-neutralization of SARS-CoV-2 variants in convalescent COVID-19 patients. We performed immunological profiling of 69 tocilizumab-treated and non-treated convalescent COVID-19 patients in total. We observed that SARS-CoV-2-specific IgG1 titers depended on disease severity but not on tocilizumab treatment. The plasma of both treated and non-treated patients infected with the ancestral variant exhibit strong neutralizing activity against the ancestral virus and the Alpha, Beta, and Delta variants of SARS-CoV-2, whereas the Gamma and Omicron viruses were less sensitive to seroneutralization. Overall, we observed that, despite the clinical benefits of short-term tocilizumab therapy in modifying the cytokine storm associated with COVID-19 infections, there were no modifications in the robustness of B cell and IgG responses to Spike antigens.

Supplementary Figure 1 from Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

Supplementary Figure Legends 1-2 from Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

Supplementary Figure 2 from Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). The pSpike/PP-sNp not only displays superior gene transfection and favorable biocompatibility in the mouse airway, but also promotes a tripartite immunity consisting of systemic, cellular, and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, immunization with pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp is a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

Aerosol lung gene therapy using non-viral delivery systems represents a credible therapeutic strategy for chronic respiratory diseases, such as cystic fibrosis (CF). Progress in CF clinical setting using the lipidic formulation GL67A has demonstrated the relevance of such a strategy while emphasizing the need for more potent gene transfer agents. In recent years, many novel non-viral gene delivery vehicles were proposed as potential alternatives to GL67 cationic lipid. However, they were usually evaluated using procedures difficult or even impossible to implement in clinical practice. In this study, a clinically-relevant administration protocol via aerosol in murine lungs was used to conduct a comparative study with GL67A. Diverse lipidic compounds were used to prepare a series of formulations inspired by the composition of GL67A. While some of these formulations were ineffective at transfecting murine lungs, others demonstrated modest-to-very-efficient activities and a series of structure-activity relationships were unveiled. Lipidic aminoglycoside derivative-based formulations were found to be at least as efficient as GL67A following aerosol delivery of a luciferase-encoding plasmid DNA. A single aerosol treatment with one such formulation was found to mediate long-term lung transgene expression, exceeding half the animal's lifetime. This study clearly supports the potential of aminoglycoside-based cationic lipids as potent GL67-alternative scaffolds for further enhanced aerosol non-viral lung gene therapy for diseases such as CF.

Aim To investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. Methods Animals from a new transgenic rat line expressing green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received at Day-12, near neural diversification, a single oral bolus of mir-320-3p or miR-375-3p, embedded in DiOleyl-Succinyl-Paromomycin (DOSP), and were further early (Day-15) or regularly (Day-30) weaned. Relevant miRNA (miR-320-3p, miR-375-3p, miR-375-5p, miR-16-5p, miR-132-3p, miR-504), polr3d , hspb6 , inflammation, enteroendocrine, and circadian clock-related mRNAs, chromatin complexes, and duodenal cell density were assayed at 8h post-inoculation and at Day-45. Results The miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p<0.05) but no long-term effects. On regular weaning, at Day-45, both miR-320-3p and 375-3p were down-regulated in the stomach, up-regulated in the hypothalamus (p<0.001) but only miR-320-3p was up-regulated in the duodenum. After early weaning, the miR-320-3p and miR-375-3p levels were down-regulated in the stomach and the duodenum, but up-regulated in the hypothalamus and the hippocampus. Combining miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the hippocampus, the miR-504 was down-regulated for both sexes, but in the brain stem, up regulated only for females, along with miR-320-3p and miR-16-5p levels. In the hypothalamus, clock levels were up regulated for both sexes. In the miR-375-3p/DOSP group, the density of enteroendocrine duodenal cells increased. The long-term effect of miR-375-3p/DOSP was more limited, according to the fourfold lower number of predicted targets than with miR-320-3p. Conclusion Addressing oral miRNA-320-3p loads to duodenal cell lineage is paving the way for the design of new therapeutics, manipulating long term consequences of early life stress.

To investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. Animals from a new transgenic rat line expressing the green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received a single oral bolus of miR-320-3p or miR-375-3p embedded in DiOleyl-Succinyl-Paromomycin (DOSP) on D-12. The pups were weaned early (D-15), or regularly (D-30). The expression of relevant miRNA, mRNAs, chromatin complexes, and duodenal cell density were assessed at 8 h post-inoculation and on D-45. The miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p < 0.05). On regular weaning, on D-45, miR-320-3p and 375-3p were found to be downregulated in the stomach and upregulated in the hypothalamus (p < 0.001), whereas miR-320-3p was upregulated in the duodenum. After early weaning, miR-320-3p and miR-375-3p were downregulated in the stomach and the duodenum, but upregulated in the hypothalamus and the hippocampus. Combination of miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the female brain stem, miR-320-3p, miR-504, and miR-16-5p levels were all upregulated. Investigating the oral miRNA-320-3p loads in the duodenal cell lineage paved the way for designing new therapeutics to avoid unexpected long-term impacts on the brain.

Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

Recently, LDH have been considered as a new kind of gene delivery system [1, 2]. In this work, we report the formation of magnesium-gallium LDH-DNA nanohybrids using the coprecipitation method. This “self assembly” approach enabled the incorporation of long DNA fragments up to 6000–8000 bp. X-ray diffraction analyses indicate a parallel orientation of DNA double helix in the interlamellar space with respect to the hydroxide sheets. The Mg/Ga molar ratio within the hydroxide layers appears to be determined by DNA macromolecules which may interact with charged complexes that form during cation hydrolysis. The presence of DNA macromolecules also inhibits the crystal growth: hydrodynamic diameter measurements revealed homogeneous populations of particles with a mean diameter ranging from 90 to 150 nm, compatible with cell penetration through endocytosis. Concerning the charge surface of this new DNA delivery system, ζ-potential measurements indicate negative values ranging from–20 to - 40 mV which suggest incomplete DNA intercalation. Yet, this small negative surface might be suitable for protecting DNA from extra-cellular degradations without preventing cell penetration.

At the end of December 2019, China notified the World Health Organization about a viral pneumonia epidemic soon to be named COVID-19, of which the infectious agent, SARS-CoV-2, was rapidly identified. Less than one year later, published phase 3 clinical trials underlined the effectiveness of vaccines utilizing hitherto unusual technology consisting in injection of the messenger RNA (m-RNA) of a viral protein. In the meantime, numerous clinical trials had failed to identify a maximally effective antiviral treatment, and mass vaccination came to be considered as the strategy most likely to put an end to the pandemic. The objective of this text is to address and hopefully answer the questions being put forward by healthcare professionals on the different anti-SARS-CoV-2 vaccines as regards their development, their modes of action, their effectiveness, their limits, and their utilization in different situations; we are proposing a report on both today's state of knowledge, and the 14 February 2021 recommendations of the French health authorities.