Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications,such as CAR T cell-based therapies.mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral-or DNA transposon-based approaches for the generation of adoptive T cells.However,limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells.In this study,we demonstrate that gold nanoparticle-mediated vapor nanobubble(VNB)photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells.Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration,while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%.Importantly,compared to electroporation,which is the reference technology for nonviral transfection of T cells,a fivefold increase in the number of transfected viable Jurkat T cells was observed.Altogether,our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells,with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

Impact of the polymer backbone chemistry on interactions of amino-acid-derived zwitterionic polymers with cells

Zwitterionic polymers are known to interact with cells and have been shown to reveal cancer cell specificity.In this work,the importance of the chemistry of the polymer backbone for the cellular specificity of amino-acid-derived polyzwitterions is demonstrated.A series of glutamic acid(Glu)-based vinyl monomers(i.e.,an acrylate,a methacrylate,an acrylamide,and a methacrylamide)were prepared and used for reversible addition-fragmentation chain-transfer(RAFT)polymerisation,yielding defined polymers with narrow size distribution(Ð<1.3).All Glu-functionalised,zwitterionic polymers revealed high cytocompatibility;however,differences in cellular association and specificity were observed.In particular,the methacrylamide-derived polymers showed high association with both,breast cancer cells and non-cancerous dendritic cells and,consequently,lack specificity.In contrast,high specificity to only breast cancer cells was observed for polyacrylates,-methacrylates,and-acrylamides.Detailed analysis of the polymers revealed differences in hydrophobicity,zeta potential,and potential side chain hydrolysis,which are impacted by the polymer backbone and might be responsible for the altered the cell association of these polymers.It is shown that a slightly negative net charge is preferred over a neutral charge to retain cell specificity.This was also confirmed by association experiments in the presence of competitive amino acid transporter substrates.The affinity of slightly negatively charged Glu-derived polymers to the xCT Glu/cystine cell membrane antiporter was found to be higher than that of neutrally charged polymers.Our results emphasise the importance of the polymer backbone for the design of cell-specific polymers.This study further highlights the potential to tailor amino-acid-derived zwitterionic materials beyond their side chain functionality.

Atm deficient zebrafish model reveals conservation of the tumour suppressor function and a role in fertility

Biallelic loss-of-function variants in ATM (Ataxia Telangiectasia Mutated) cause Ataxia Telangiectasia (AT), a rare disorder associated with cerebellar degeneration and ataxia, cancer predisposition, infertility, growth retardation, etc. ATM is a phosphoinositide 3-kinase-related kinase (PIKK) with a role in DNA repair and maintenance of genome stability. Studying a multisystem genetic disease like AT requires animal models to ascertain its pathogenesis at the level of tissues, organs and the organism. Due to its small size, cheap maintenance, large progeny, rapid development and initial transparency, zebrafish (Danio rerio) is an increasingly popular vertebrate model organism, suitable for genetic modifications and large-scale in vivo therapeutic screens as embryos are chemically permeable to small compounds. Currently, no zebrafish model for AT exists.1 We generated atm knock-outs through CRIPSR-Cas9 mutagenesis. We show that atm conserved its function as a tumour suppressor gene and is involved in gametogenesis and fertility. Therefore, this mutant is of great value for further studies investigating the role of atm in reproduction and tumorigenesis.

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors

Several obstacles to the production,expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy.In the context of HSCT,delayed naïve T-cell recovery contributes to poor outcomes.A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors(HTLPs),allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus.However,it is challenging to produce HTLPs in the high numbers required to meet clinical needs.Here,we found that adding tumor necrosis factor alpha(TNFα)to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival.This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy(including CAR T-cell therapy).