Non-viral delivery of RNA for therapeutic T cell engineering.

Adoptive T cell transfer has shown great success in treating blood cancers, resulting in a growing number of FDA-approved therapies using chimeric antigen receptor (CAR)-engineered T cells. However, the effectiveness of this treatment for solid tumors is still not satisfactory, emphasizing the need for improved T cell engineering strategies and combination approaches. Currently, CAR T cells are mainly manufactured using gammaretroviral and lentiviral vectors due to their high transduction efficiency. However, there are concerns about their safety, the high cost of producing them in compliance with current Good Manufacturing Practices (cGMP), regulatory obstacles, and limited cargo capacity, which limit the broader use of engineered T cell therapies. To overcome these limitations, researchers have explored non-viral approaches, such as membrane permeabilization and carrier-mediated methods, as more versatile and sustainable alternatives for next-generation T cell engineering. Non-viral delivery methods can be designed to transport a wide range of molecules, including RNA, which allows for more controlled and safe modulation of T cell phenotype and function. In this review, we provide an overview of non-viral RNA delivery in adoptive T cell therapy. We first define the different types of RNA therapeutics, highlighting recent advancements in manufacturing for their therapeutic use. We then discuss the challenges associated with achieving effective RNA delivery in T cells. Next, we provide an overview of current and emerging technologies for delivering RNA into T cells. Finally, we discuss ongoing preclinical and clinical studies involving RNA-modified T cells.

Delivery of macromolecules in unstimulated T cells by photoporation with polydopamine nanoparticles.

Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500 kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400 nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150 nm and 250 nm PDNPs retained their propensity to become activated, whereas those photoporated with 400 nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage.

Photothermal nanofibres enable safe engineering of therapeutic cells.

Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR-Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.

Evaluation of tenogenic differentiation potential of selected subpopulations of human adipose-derived stem cells.

Identification of a suitable cell source and bioactive agents guiding cell differentiation towards tenogenic phenotype represents a prerequisite for advancement of cell-based therapies for tendon repair. Human adipose-derived stem cells (hASCs) are a promising, yet intrinsically heterogenous population with diversified differentiation capacities. In this work, we investigated antigenically-defined subsets of hASCs expressing markers related to tendon phenotype or associated with pluripotency that might be more prone to tenogenic differentiation, when compared to unsorted hASCs. Subpopulations positive for tenomodulin (TNMD+ hASCs) and stage specific early antigen 4 (SSEA-4+ hASCs), as well as unsorted ASCs were cultured up to 21 days in basic medium or media supplemented with TGF-ß3 (10 ng/ml), or GDF-5 (50 ng/ml). Cell response was evaluated by analysis of expression of tendon-related markers at gene level and protein level by real time RT-PCR, western blot, and immunocytochemistry. A significant upregulation of scleraxis was observed for both subpopulations and unsorted hASCs in the presence of TGF-ß3. More prominent alterations in gene expression profile in response to TGF-ß3 were observed for TNMD+ hASCs. Subpopulations evidenced an increased collagen III and TNC deposition in basal medium conditions in comparison with unsorted hASCs. In the particular case of TNMD+ hASCs, GDF-5 seems to influence more the deposition of TNC. Within hASCs populations, discrete subsets could be distinguished offering varied sensitivity to specific biochemical stimulation leading to differential expression of tenogenic components suggesting that cell subsets may have distinctive roles in the complex biological responses leading to tenogenic commitment to be further explored in cell based strategies for tendon tissues.

Bi-directional modulation of cellular interactions in an in vitro co-culture model of tendon-to-bone interface.

OBJECTIVES: This work aimed at studying in vitro interactions between human tendon-derived cells (hTDCs) and pre-osteoblasts (pre-OBs) that may trigger a cascade of events involved in enthesis regeneration. MATERIALS AND METHODS: The effect of 5 osteogenic medium (OM) conditions over the modulation of hTDCs and pre-OBs towards the tenogenic and osteogenic phenotypes, respectively, was studied. Three different medium conditions were chosen for subsequently establishing a direct co-culture system in order to study the expression of bone, tendon and interface-related markers. RESULTS: A higher matrix mineralization and ALP activity was observed in co-cultures in the presence of OM. Higher transcription levels of bone- (ALPL, RUNX2, SPP1) and interface-related genes (ACAN, COMP) were found in co-cultures. The expression of aggrecan was influenced by the presence of OM and cell-cell interactions occurring in co-culture. CONCLUSIONS: The present work assessed both the influence of OM on cell phenotype modulation and the importance of co-culture models while promoting cell-cell interactions and the exchange of soluble factors in triggering an interface-like phenotype to potentially modulate enthesis regeneration.

Tendon explant cultures to study the communication between adipose stem cells and native tendon niche.

Poor clinical outcomes of tendon repair, together with limited regenerative capacity of the tissue, have triggered the search for alternative regenerative medicine strategies. Human adipose-derived stem cells (hASCs) are being investigated as a promising cell source in contributing for tendon repopulation and reconstruction. However, the mechanisms involved in a potential beneficial effect in tendon regeneration are still to be uncovered. To gain further insights on the bi-directional crosstalk occurring between stem cells and the native tendon niche, it was used an indirect (trans-well) system for co-culturing human tendon explants and hASCs. The maintenance of tissue architecture was studied up to 14 days by histological techniques. The secretion of MMPs was evaluated at day 3. The behavior of hASCs was assessed regarding cell elongation and extracellular matrix (ECM) production. The paracrine communication enhanced collagenolytic activity of MMPs in co-cultures at day 3, in comparison to hASCs alone or tendon explants alone, suggesting that ECM remodeling is triggered early in culture. Moreover, hASCs were spontaneously more elongated in co-cultures and the deposition of collagen type III and tenascin-C by hASCs in co-culture was observed at a lower extent after 7 days, in comparison to hASCs alone, being lately recovered at day 14. Overall, explant co-cultures established herein may constitute a tool for replicating the first steps in tendon healing and help uncovering the bi-directional communication occurring between hASCs and the native tendon niche.

Monocyte Chemoattractant Protein-Induced Protein 1 (MCPIP1) Enhances Angiogenic and Cardiomyogenic Potential of Murine Bone Marrow-Derived Mesenchymal Stem Cells.

The current evidence suggests that beneficial effects of mesenchymal stem cells (MSCs) toward myocardial repair are largely due to paracrine actions of several factors. Although Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is involved in the regulation of inflammatory response, apoptosis and angiogenesis, whether MCPIP1 plays any role in stem cell-induced cardiac repair has never been examined. By employing retroviral (RV)-transduced overexpression of MCPIP1, we investigated the impact of MCPIP1 on viability, apoptosis, proliferation, metabolic activity, proteome, secretome and differentiation capacity of murine bone marrow (BM) - derived MSCs. MCPIP1 overexpression enhanced angiogenic and cardiac differentiation of MSCs compared with controls as indicated by elevated expression of genes accompanying angiogenesis and cardiomyogenesis in vitro. The proangiogenic activity of MCPIP1-overexpressing MSCs (MCPIP1-MSCs) was also confirmed by increased capillary-like structure formation under several culture conditions. This increase in differentiation capacity was associated with decreased proliferation of MCPIP1-MSCs when compared with controls. MCPIP1-MSCs also expressed increased levels of proteins involved in angiogenesis, autophagy, and induction of differentiation, but not adverse inflammatory agents. We conclude that MCPIP1 enhances endothelial and cardiac differentiation of MSCs. Thus, modulating MCPIP1 expression may be a novel approach useful for enhancing the immune-regulatory, anti-apoptotic, anti-inflammatory and regenerative capacity of BM-derived MSCs for myocardial repair and regeneration of ischemic tissues.