An ionizable supramolecular dendrimer nanosystem for effective siRNA delivery with a favorable safety profile

Gene therapy using small interfering RNA(siRNA)is emerging as a novel therapeutic approach to treat various diseases.However,safe and efficient siRNA delivery still constitutes the major obstacle for clinical implementation of siRNA therapeutics.Here we report an ionizable supramolecular dendrimer vector,formed via self-assembly of a small amphiphilic dendrimer,as an effective siRNA delivery system with a favorable safety profile.By virtue of the ionizable tertiary amine terminals,the supramolecular dendrimer has a low positively charged surface potential and no notable cytotoxicity at physiological pH.Nonetheless,this ionizable feature imparted sufficient surface charge to the supramolecular dendrimer to enable formation of a stable complex with siRNA via electrostatic interactions.The resulting siRNA/dendrimer delivery system had a surface charge that was neither neutral,thus avoiding aggregation,nor too high,thus avoiding cytotoxicity,but was sufficient for favorable cellular uptake and endosomal release of the siRNA.When tested in different cancer cell lines and patient-derived cancer organoids,this dendrimer-mediated siRNA delivery system effectively silenced the oncogenes Myc and Akt2 with a potent antiproliferative effect,outperforming the gold standard vector,Lipofectamine 2000.Therefore,this ionizable supramolecular dendrimer represents a promising vector for siRNA delivery.The concept of supramolecular dendrimer nanovectors via self-assembly is new,yet easy to implement in practice,offering a new perspective for supramolecular chemistry in biomedical applications.

High-throughput multiplexed fluorescence-activated droplet sorting

Fluorescence-activated droplet sorting(FADS)is one of the most important features provided by droplet-based microfluidics.However,to date,it does not allow to compete with the high-throughput multiplexed sorting capabilities offered by flow cytometery.Here,we demonstrate the use of a dielectrophoretic-based FADS,allowing to sort up to five different droplet populations simultaneously.Our system provides means to select droplets of different phenotypes in a single experimental run to separate initially heterogeneous populations.Our experimental results are rationalized with the help of a numerical model of the actuation of droplets in electric fields providing guidelines for the prediction of sorting designs for upscaled or downscaled microsystems.

Molecular mechanisms of liver carcinogenesis related to metabolic syndrome

Global prevalence of non-alcoholic fatty liver disease(NAFLD)and of NAFLD-hepatocellular carcinoma(HCC)is estimated to grow in the next years.The burden of NAFLD and the evidence that NAFLD-HCC arises also in noncirrhotic patients,explain the urgent need of a better characterization of the molecular mechanisms involved in NAFLD progression.Obesity and diabetes cause a chronic inflammatory state which favors changes in serum cytokines and adipokines,an increase in oxidative stress,DNA damage,and the activation of multiple signaling pathways involved in cell proliferation.Moreover,a role in promoting NAFLD-HCC has been highlighted in the innate and adaptive immune system,dysbiosis,and alterations in bile acids metabolism.Several dietary,genetic,or combined mouse models have been used to study nonalcoholic steatohepatitis(NASH)development and its progression to HCC,but models that fully recapitulate the biological and prognostic features of human NASH are still lacking.In humans,four single nucleotide polymorphisms(PNPLA3,TM6SF2,GCKR,and MBOAT7)have been linked to the development of both NASH and HCC in cirrhotic and non-cirrhotic patients,whereas HSD17B13 polymorphism has a protective effect.In addition,higher rates of somatic ACVR2A mutations and a novel mutational signature have been recently discovered in NASH-HCC patients.The knowledge of the molecular pathogenesis of NAFLD-HCC will be helpful to personalized screening programs and allow for primary and secondary chemopreventive treatments for NAFLD patients who are more likely to progress to HCC.

Ypel5 regulates liver development and function in zebrafish

YPEL5 is a member of the Yippee-like(YPEL)gene family that is evolutionarily conserved in eukaryotic species.To date,the physiological function of YPEL5 has not been assessed due to a paucity of genetic animal models.Here,using CRISPR/Cas9-mediated genome editing,we generated a stable ypel5^(−/−)mutant zebrafish line.Disruption of ypel5 expression leads to liver enlargement associated with hepatic cell proliferation.Meanwhile,hepatic metabolism and function are dysregulated in ypel5^(−/−)mutant zebrafish,as revealed by metabolomic and transcriptomic analyses.Mechanistically,Hnf4a is identified as a crucial downstream mediator that is positively regulated by Ypel5.Zebrafish hnf4a overexpression could largely rescue ypel5 deficiencyinduced hepatic defects.Furthermore,PPARαsignaling mediates the regulation of Hnf4a by Ypel5 through directly binding to the transcriptional enhancer of the Hnf4a gene.Herein,this work demonstrates an essential role of Ypel5 in hepatocyte proliferation and function and provides the first in vivo evidence for a physiological role of the ypel5 gene in vertebrates.

Therapeutic siRNA:state of the art

RNA interference(RNAi)is an ancient biological mechanism used to defend against external invasion.It theoretically can silence any disease-related genes in a sequence-specific manner,making small interfering RNA(siRNA)a promising therapeutic modality.After a two-decade journey from its discovery,two approvals of siRNA therapeutics,ONPATTRO®(patisiran)and GIVLAARI™(givosiran),have been achieved by Alnylam Pharmaceuticals.Reviewing the long-term pharmaceutical history of human beings,siRNA therapy currently has set up an extraordinary milestone,as it has already changed and will continue to change the treatment and management of human diseases.It can be administered quarterly,even twice-yearly,to achieve therapeutic effects,which is not the case for small molecules and antibodies.The drug development process was extremely hard,aiming to surmount complex obstacles,such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity,stability,specificity and potential off-target effects.In this review,the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed.All clinically explored and commercialized siRNA delivery platforms,including the GalNAc(N-acetylgalactosamine)–siRNA conjugate,and their fundamental design principles are thoroughly discussed.The latest progress in siRNA therapeutic development is also summarized.This review provides a comprehensive view and roadmap for general readers working in the field.

Amphiphilic Dendrimer Vectors for RNA Delivery:State-of-the-Art and Future

Dendrimers,a special family of polymers,are particularly promising materials for various biomedical applications by virtue of their well-defined dendritic structure and cooperative multivalency.Specifically,in this Account,we present state-of-the-art amphiphilic dendrimers for nucleic acid delivery.Ribonucleic acid(RNA)molecules are fast becoming an important drug modality,particularly since the recent success of mRNA vaccines against COVID-19.Notably,RNA therapeutics offer the unique opportunity to treat diseases at the gene level and address“undruggable”targets.However,RNA therapeutics are not stable and have poor bioavailability,imposing the need for their protection and safe delivery by vectors to the sites-of-action to allow the desired therapeutic effects.Currently,the two most advanced nonviral vectors are based on lipids and polymers,with lipid vectors primarily exploiting the membrane-fusion mechanism and polymer vectors mainly endocytosis-mediated delivery.Notably,only lipid vectors have been advanced through to their clinical use in the delivery of,for example,the first siRNA drug and the first mRNA vaccine.The success of lipid vectors for RNA delivery has motivated research for further innovative materials as delivery vectors.Specifically,we have pioneered lipid/dendrimer conjugates,referred to as amphiphilic dendrimers,for siRNA delivery with the view to harnessing the delivery advantages of both lipid and polymer vectors while enjoying the unique structural features of dendrimers.These amphiphilic dendrimer vectors are lipid/dendrimer hybrids and are thus able to mimic lipid vectors and exploit membranefusion-mediated delivery,while simultaneously retaining the multivalent properties of polymer vectors that allow endocytosis-based delivery.In addition,they have precisely controllable and stable nanosized chemical structures and offer nanotechnology-based delivery.Effective amphiphilic dendrimer vectors share two important elements:chemical hydrophilic entities to bind RNA and RNA complex-stabilizing hydrophobicity.These two combined features allow the encapsulation of RNA within a stable complex before its release into the cytosol following endocytosis.This hydrophilic/hydrophobic balance permitted by the structural features of amphiphilic dendrimers plays a determining role in RNA delivery success.In this Account,we provide a conceptual overview of this exciting field with the latest breakthroughs and key advances in the design of amphiphilic dendrimers for the delivery of siRNA and mRNA.Specifically,we start with a short introduction to siRNA-and mRNA-based therapeutics and their delivery challenges.We then outline the pioneering and representative studies on amphiphilic dendrimer vectors to highlight their historical development and promising features that offer to facilitate the once challenging RNA delivery.We conclude by offering perspectives for the future of amphiphilic dendrimer vectors for nucleic acid delivery in general.

S1P: the elixir of life for naive T cells

During their life,T cells are constantly circulating throughout the body.Sphingosine-1-phosphate(S1P),a sphingolipid metabolite,triggers T cell egress from the thymus and secondary lymphoid organs(SLO)into the lymph and the blood.This phenomenon is dependent on the S1P gradient between lymphoid organs(including thymus and SLO)and the lymphatic and blood vessels,which exhibit low,intermediate and high S1P levels,respectively.T cells follow the S1P gradient via the engagement of the G-protein-coupled receptor S1P receptor 1(S1P1)expressed on T cells.1 S1P production is regulated by various sphingolipid-metabolizing enzymes.The sphingosine kinases 1 and 2(SKs),encoded by Sphk1 and Sphk2,phosphorylate the sphingosine(Sph)into S1P.Conversely,S1P can be dephosphorylated to Sph by S1P phosphatases 1 and 2,encoded by Sgpp1 and Sgpp2.Alternatively,S1P can be irreversibly degraded by the S1P Lyase(SPL),encoded by Sgpl1,into phosphoethanolamine and hexadecenal.

Systemic treatment of hepatocellular carcinoma:the times they are a-changin’

For a decade,sorafenib remained the only drug approved for the treatment of advanced hepatocellular carcinoma(HCC)due to the failure of several phase III trials in which alternative angiogenesis inhibitors were tested(1).In recent years,the field of systemic treatments of HCC has evolved quickly and a recent position paper of the European Association for the Study of the Liver has well described the current advances in the systemic treatment of advanced HCC as well as the unmet needs in the field(1).First,lenvatinib was approved as an alternative to sorafenib in the first-line setting,and,thus,regorafenib,cabozantinib and ramucirumab as second-line treatments for patients who progressed on sorafenib(1).