Biology subjects

Ryaykenen, T.

Publications and source records attributed to Ryaykenen, T..

4 recordsLinked to original sources

bioRxiv2025

Cryopreservation of siRNA-treated cells is feasible

Cryopreservation is a routine step in the manufacturing process of adoptive cell therapies, providing critical logistic flexibility. RNAi-based therapies are increasingly being explored as enhancers or modulators of adoptive cell therapies. However, the impact of cryopreservation on cells treated with RNAi-based therapies has not been investigated before. In this study, we addressed this knowledge gap by examining silencing efficacy in siRNA-treated cells that undergo cryopreservation. Our findings demonstrate that silencing in cryopreserved cells is comparable to that in cells maintained continuously in culture. Moreover, we found that the duration of siRNA exposure plays a significant role in cells that later undergo cryopreservation, with extended exposure improving silencing efficiency. However, this effect diminishes at higher siRNA concentrations. Additionally, we showed that siRNA treatment is feasible at low temperatures (2-8{degrees}C), and siRNA-treated cells can be cryopreserved for extended periods (at least one month) without loss of efficacy. Furthermore, we demonstrated the feasibility of cryopreserving siRNA-treated primary cells, including those resembling leukapheresis material. Our work establishes the feasibility of integrating siRNA treatments into current manufacturing processes for adoptive cell therapies.

bioengineering↗

bioRxiv2025

miRNA Mimic Optimization: Chemical Structure - Activity - Targetome Relationship to Engineer Selective Anti-Tumor Immunity in T cells

MicroRNA (miRNA) mimic therapies act through a broad and complex targetome in disease contexts. However, both the composition of these targetomes and the impact of chemical modifications on them remain poorly understood. In this study, we investigate eight fully chemically modified miRNA scaffolds across three miRNA sequences in a model immune disorder, graft-versus-host-disease (GvHD), which is an off-tumor effect of allogeneic T cell therapy. We demonstrate that conventional silencing assays fail to predict the functional performance of miRNA mimics in GVHD and graft-versus-leukemia (GvL), the on-tumor effect of allogeneic T cells. Moreover, we find that chemical scaffolds influence the duration of silencing mediated by miRNA mimics. We identify a miR-374b version as a lead miRNA candidate that not only inhibits GVHD but also enhances GVL--an unprecedented and desired improvement in on-versus-off-tumor selectivity of clinical relevance. Further analysis reveals distinct responder and non-responder groups to miR-374b therapy. While responders exhibit significant transcriptomic shifts, non-responders show virtually no changes, despite intact miRNA pathway function, underscoring a strong correlation between transcriptome reprogramming and therapeutic efficacy. RNA sequencing reveals that miR-374b acts via metabolic reprogramming of T cells and induces partially distinct transcriptional programs in GVHD and GVL. Collectively, our findings demonstrate that fully chemically modified miRNA mimic therapies are feasible to rewrite the T-cell transcriptome, profoundly improving on-versus-off-tumor specificity in T-cell-mediated cancer therapies.

bioengineering↗

bioRxiv2024

Structure - silencing duration relationships in RNAi medicines in rapidly dividing cells

RNA interference (RNAi)-based medicines offer precise targeting of virtually any transcript, making them an appealing new drug class for addressing unmet needs in immune-oncological applications. While RNAi therapies show exceptional duration of effect in non-dividing cells, their efficacy in rapidly dividing cells, crucial for immune-oncology, remains largely unexplored. Unlike in non-dividing cells, full chemical modification in rapidly dividing cells has not consistently extended silencing duration, according to limited data available. In this study, we investigated key factors affecting the duration of effect for three main types of RNAi-based therapeutics (siRNA, miRNA mimics, and miRNA inhibitors) in rapidly dividing cancer and immune cells. Saturation of intracellular depots by multiple loading doses, a common strategy to prolong silencing duration in non-dividing hepatocytes, had minimal impact on siRNA duration of effect in rapidly dividing cells. However, modifying the antisense strand with a 5-(E)-vinylphosphonate (5-VP) to protect siRNAs from exonucleases and enhance AGO2 binding significantly extended siRNA silencing duration to over 30 days both in vitro and in vivo. For miRNA mimics, extensive stabilization of the antisense strand with phosphorothioates was not effective and led to reduced potency and silencing duration. Interestingly, a shorter duplex region commonly seen in therapeutic siRNAs partially rescued duration of silencing in miRNA mimics with extended phosphorothioate modifications. On the other hand, miRNA inhibitors demonstrated robust reversal of miRNA activity for an impressive 25 days in cancer cell lines. Our findings enable the rational design of the chemical architecture and administration regimens of RNAi-based therapies in oncology and immunology.

pharmacology and toxicology↗

bioRxiv2023

Systematic optimization of siRNA productive uptake into resting and activated T cells ex vivo

RNA-based medicines are ideally suited for precise modulation of T cell phenotypes in anti-cancer immunity, in autoimmune diseases and for ex vivo modulation of T-cell-based therapies. Therefore, understanding productive siRNA uptake to T cells is of particular importance. Most studies used unmodified siRNAs or commercially available siRNA with undisclosed chemical modifications patterns to show functionality in T cells. Despite being an active field of research, robust siRNA delivery to T cells still represents a formidable challenge. Therefore, a systematic approach is needed to further optimize and understand productive siRNA uptake pathways to T cells. Here we compared conjugate-mediated and nanoparticle-mediated delivery of siRNAs to T cells in the context of fully chemically modified RNA constructs. We showed that lipid-conjugate-mediated delivery outperforms lipid-nanoparticle-mediated and extracellular-vesicle-mediated delivery in activated T cells ex vivo. Yet, ex vivo manipulation of T cells without the need of activation is of great therapeutic interest for CAR-T, engineered TCR-T and allogeneic donor lymphocyte applications. We are first to report productive siRNA uptake into resting T cells using lipid-conjugate mediated delivery. Interestingly, we observed strong dependence of silencing activity on lipid-conjugate-identity in resting T cells but not in activated T cells. This phenomenon is consistent with our early uptake kinetics data. Lipid-conjugates also enabled delivery of siRNA to all mononuclear immune cell types, including both lymphoid and myeloid lineages. These findings are expected to be broadly applicable for ex vivo modulation of immune cell therapies.

molecular biology↗