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

Non-invasive cell-tracking methods for adoptive T cell therapies

Adoptive T cell therapies (ACT) have demonstrated groundbreaking results in blood cancers and melanoma. Nevertheless, their significant cost, the occurrence of severe adverse events, and their poor performance in solid tumors are important hurdles hampering more widespread applicability. In vivo cell-tracking allows instantaneous and non-invasive monitoring of the distribution, tumor homing, persistence, and redistribution to other organs of infused T cells in patients. Furthermore, cell-tracking could aid in the clinical management of patients, allowing the detection of non-responders or severe adverse events at an early stage. This review provides a concise overview of the main principles and potential of cell-tracking, followed by a discussion of the clinically relevant labeling strategies and their application in ACT

Non-viral siRNA delivery to T cells : challenges and opportunities in cancer immunotherapy

T lymphocytes are the major drivers of antitumor immunity. The recent clinical success of adoptive T cell therapies and immune checkpoint inhibitors has demonstrated the strength of modulating T cell function in fighting cancer. Nonetheless, a significant fraction of patients remain unresponsive largely due to the immunosuppressive tumor environment that blunts T cell activity. Small interfering RNAs (siRNAs) offer the potential to sequence-specifically silence the expression of negative regulator genes in T cells in a transient manner, thereby releasing the block on anti-tumor responses. Despite the current focus on small molecule- and antibody-based immune checkpoint inhibitors as well as T cell-directed delivery of mRNA and genome editing machinery, the application of siRNA involves important clinical advantages. The recent surge of adoptive cell therapies and development of new and potent delivery approaches has enabled efficient siRNA delivery to T cells both ex vivo and in vivo. As such, siRNA molecules have a newfound potential to improve the proliferation, survival, tumor infiltration and potency of T cells in cancer immunotherapy. In this review, we briefly discuss the extracellular and intracellular delivery hurdles associated with siRNA therapy, in particular with regard to T cell targeting. We provide a timely and comprehensive overview of current and emerging delivery technologies used for siRNA transfection, discussing their strengths and weaknesses from a clinical as well as a manufacturing point-of-view. Finally, we critically review the current status and new potential avenues for modulating T cell function in cancer immunotherapy using siRNA

Cationic amphiphilic drugs boost the lysosomal escape of small nucleic acid therapeutics in a nanocarrier-dependent manner

Small nucleic acid (NA) therapeutics, such as small interfering RNA (siRNA), are generally formulated in nanoparticles (NPs) to overcome the multiple extra- and intracellular barriers upon in vivo administration. Interaction with target cells typically triggers endocytosis and sequesters the NPs in endosomes, thus hampering the pharmacological activity of the encapsulated siRNAs that occurs in the cytosol. Unfortunately, for most state-of-the-art NPs, endosomal escape is largely inefficient. As a result, the bulk of the endocytosed NA drug is rapidly trafficked toward the degradative lysosomes that are considered as a dead end for siRNA nanomedicines. In contrast to this paradigm, we recently reported that cationic amphiphilic drugs (CADs) could strongly promote functional siRNA delivery from the endolysosomal compartment via transient induction of lysosomal membrane permeabilization. However, many questions still remain regarding the broader applicability of such a CAD adjuvant effect on NA delivery. Here, we report a drug repurposing screen (National Institutes of Health Clinical Collection) that allowed identification of 56 CAD adjuvants. We furthermore demonstrate that the CAD adjuvant effect is dependent on the type of nanocarrier, with NPs that generate an appropriate pool of decomplexed siRNA in the endolysosomal compartment being most susceptible to CAD-promoted gene silencing. Finally, the CAD adjuvant effect was verified on human ovarian cancer cells and for antisense oligonucleotides. In conclusion, this study strongly expands our current knowledge on how CADs increase the cytosolic release of small NAs, providing relevant insights to more rationally combine CAD adjuvants with NA-loaded NPs for future therapeutic applications

MIF inhibition interferes with the inflammatory and T cell-stimulatory capacity of NOD macrophages and delays autoimmune diabetes onset

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression.status: publishe

MIF inhibition interferes with the inflammatory and T cell-stimulatory capacity of NOD macrophages and delays autoimmune diabetes onset.

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression

Hydrogel-Induced Cell Membrane Disruptions Enable Direct Cytosolic Delivery of Membrane-Impermeable Cargo

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines

Hydrogel‐induced cell membrane disruptions enable direct cytosolic delivery of membrane‐impermeable cargo

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T-1-weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines

Quantification and visualization of MIF and CD74-expressing cells in the pancreas of NOD mice.

<p>Quantification of the relative MIF and CD74 mRNA levels in the total pancreas of control and NOD mice by means of real-time reverse transcription PCR (RT-PCR) (Mean ± SEM; n = 4–5) (A &B) (*: p-values ≤ 0.05). (C) Pancreatic sections of NOD mice were stained for insulin (white), MIF (green), F4/80 (red) and DNA by DAPI (blue) and analyzed with confocal microscopy at ×40 original magnification. Cells featuring a co-localization of MIF and the macrophage marker F4/80 are indicated by the white arrows. (D) Confocal microscope image of pancreatic sections of NOD mice were stained for insulin (white), CD74 (green), F4/80 (red) and DNA by DAPI (blue) (×40 original magnification). The images depicted shows one representative microphotograph documenting the presence of multiple MIF⁺- or CD74⁺ cells within the pancreatic islets of 8-week-old NOD mice. Similar results were obtained with 12-week-old acutely diabetic NOD mice (not shown). (E) Quantification of the percentage of F4/80⁺CD11b⁺ macrophages within the live leukocyte gate (mean ± SEM; n = 4–5) (*: p-values ≤ 0.05). (F) Quantification of the percentage of CD74⁺ cells within the F4/80⁺CD11b⁺ macrophage population as determined on homogenized pancreas samples by flow cytometry (mean ± SEM; n = 4–5).</p