Biology subjects

Cooper, D. A.

Publications and source records attributed to Cooper, D. A..

7 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↗

bioRxiv2024

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ER) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ER ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ER breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29s activities in breast cancer cells harboring Y537S ER. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

cancer biology↗

bioRxiv2024

Intracellular pocket conformations determine signaling through the μ opioid receptor

It has been challenging to determine how a ligand that binds to a receptor activates downstream signaling pathways and to predict the strength of signaling. The challenge is compounded by functional selectivity, in which a single ligand binding to a single receptor can activate multiple signaling pathways at different levels. Spectroscopic studies show that in the largest class of cell surface receptors, 7 transmembrane receptors (7TMRs), activation is associated with ligand-induced shifts in the equilibria of intracellular pocket conformations in the absence of transducer proteins. We hypothesized that signaling through the opioid receptor, a prototypical 7TMR, is linearly proportional to the equilibrium probability of observing intracellular pocket conformations in the receptor-ligand complex. Here we show that a machine learning model based on this hypothesis accurately calculates the efficacy of both G protein and {beta}-arrestin-2 signaling. Structural features that the model associates with activation are intracellular pocket expansion, toggle switch rotation, and sodium binding pocket collapse. Distinct pathways are activated by different arrangements of the ligand and sodium binding pockets and the intracellular pocket. While recent work has categorized ligands as active or inactive (or partially active) based on binding affinities to two conformations, our approach accurately computes signaling efficacy along multiple pathways.

biophysics↗

bioRxiv2021

NOVA2 regulates neural circRNA biogenesis

Circular RNAs (circRNAs) are highly expressed in the brain and their expression increases during neuronal differentiation. The factors regulating circRNAs in the developing mouse brain are unknown. NOVA1 and NOVA2 are neural-enriched RNA-binding proteins with well-characterized roles in alternative splicing. Profiling of circRNAs from RNA-seq data revealed that global circRNA levels were reduced in embryonic cortex of Nova2 but not Nova1 knockout mice. Analysis of isolated inhibitory and excitatory cortical neurons lacking NOVA2 revealed an even more dramatic reduction of circRNAs and establish a widespread role for NOVA2 in enhancing circRNA biogenesis. To investigate the cis-elements controlling NOVA2-regulation of circRNA biogenesis, we generated a backsplicing reporter based on the Efnb2 gene. We found that NOVA2-mediated backsplicing of circ-Efnb2 was impaired when YCAY clusters located in flanking introns were removed. CLIP and additional reporter analysis demonstrated the importance of NOVA2 binding sites located in both flanking introns of circRNA loci. NOVA2 is the first RNA-binding protein identified to globally promote circRNA biogenesis in the developing brain.

genomics↗

bioRxiv2017

Global accumulation of circRNAs during aging in Caenorhabditis elegans

Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5{acute} and 3{acute} ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated 1,166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by qRT-PCR. The age-accumulation of circRNAs was not accompanied by increased expression of linear RNAs from the same host genes. We attribute the global scale of circRNA age-accumulation to the high composition of postmitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine if there are functional consequences of circRNA accumulation during aging.

genomics↗