Biology subjects

Yarovinsky, T. O.

Publications and source records attributed to Yarovinsky, T. O..

4 recordsLinked to original sources

bioRxiv2026

Disrupting miR-466l-3p and HuR Cooperation with Target Site Blockers Reveals a Therapeutic Strategy to Destabilize mRNA Transcripts

MicroRNAs (miRNAs) typically regulate gene expression by promoting mRNA degradation, but select miRNAs, such as miR-466l-3p (miR-466), can instead stabilize transcripts in coordination with RNA-binding proteins (RBPs) like HuR. We identify conserved AU-rich elements (cAREs) within the 3'UTRs of IL-17A, GM-CSF, and IL-23A as critical cis-regulatory binding sites where miR-466 facilitates HuR recruitment to promote mRNA stability. Using site-directed mutagenesis, RNA pulldown, and MS2-TRAP assays to capture miRNA-mRNA complexes, we demonstrate that HuR binding depends on prior engagement by miR-466. Disrupting this interaction with rationally designed Target Site Blockers (TSBs) oligonucleotides destabilizes target mRNAs and suppresses cytokine expression in vitro and in vivo. TSBs directed against IL-17A, GM-CSF, and IL-23A selectively blocked miR-466 binding, reduced transcript stability, and lowered cytokine production without affecting unrelated mRNAs. In murine models of LPS-induced inflammation, psoriasis, and autoimmunity, TSBs exhibited therapeutic efficacy and cytokine specificity, outperforming monoclonal antibodies in some settings. Phosphorothioate-modified TSBs enabled systemic delivery and retained activity in human T cells, underscoring translational potential. Similar to antisense oligonucleotides, TSBs trigger RNase H1-mediated degradation while also blocking miRNA-mRNA interactions. These findings establish miR-466-HuR cooperation as a therapeutically targetable axis through TSBs without affecting global miRNA function. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/709388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1a87bceorg.highwire.dtl.DTLVardef@5824b2org.highwire.dtl.DTLVardef@d14f6eorg.highwire.dtl.DTLVardef@1b5c033_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TEXTBOXMechanism of TSB-mediated disruption of cooperative miRNA-HuR-dependent mRNA stabilizationA: In the canonical model, destabilizing miRNAs (e.g., miR-16) bind to their target sites within the 3'UTR, recruiting the RNA-induced silencing complex (miRISC) to promote mRNA decay or translational repression. B: In contrast, a newly identified class of miRNAs--stabilizing miRNAs (E-miRNAs), such as miR-466l-3p--bind to specific target sequences within AU-rich elements (AREs) in the 3'UTR. This binding facilitates cooperative recruitment of the RNA-binding protein HuR (ELAVL1), resulting in enhanced mRNA stability and/or translation. C: Target site blockers (TSBs) designed to occlude miRNA-binding sites competitively inhibit miRISC loading, thereby disrupting HuR engagement and reversing stabilization. This selective disruption leads to transcript-specific mRNA destabilization without affecting global miRNA function. C_TEXTBOX

molecular biology↗

bioRxiv2023

Immune modulation of innate and adaptive responses restores immune surveillance and establishes anti-tumor immunological memory

Current immunotherapies have proven effective in strengthening anti-tumor immune responses but constant opposing signals from tumor cells and surrounding microenvironment eventually lead to immune escape. We hypothesize that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system will provide a robust and long-term anti-tumor effect by creating immunological memory against the tumor. To achieve this, we developed CARG-2020, a virus-like-vesicle (VLV). It is a genetically modified and self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three transgenes: 1) the pleiotropic antitumor cytokine IL-12 in which the subunits (p35 and p40) are tethered together; 2) the extracellular domain (ECD) of the pro-tumor IL-17RA, which can serve as a dominant negative antagonist; and 3) shRNA for PD-L1. Using a mouse model of ovarian cancer, we demonstrate the oncolytic effect and immune modulatory capacities of CARG-2020. By enhancing IL-12 and blocking IL-17 and PD-L1, CARG-2020 successfully reactivates immune surveillance by promoting M1 instead of M2 macrophage differentiation, inhibiting MDSC expansion, and establishing a potent CD8+ T cell mediated anti-tumoral response. Furthermore, we demonstrate that this therapeutic approach provides tumor-specific and long-term protection preventing the establishment of new tumors. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance the anti-tumor response and to prevent recurrence.

immunology↗

bioRxiv2021

Distinct Hypoxia-induced Translational Profiles of Embryonic and Adult-derived Macrophages

Tissue homeostasis and repair are orchestrated by resident and newly recruited macrophages that alter their gene expression program in response to changes in tissue microenvironment. Embryonic macrophages, such as fetal liver derived macrophages (FLDM) seed the organs, including heart and lung during embryonic development and persist throughout the adult lifetime, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation. Transcriptome analyses of FLDM and BMDM identified differences between them at the level of RNA expression, which correlates imperfectly with protein levels. Post-transcriptional regulation by microRNAs (miRNAs) and RNA-binding proteins determines mRNA stability and translation rate and may override transcriptional cues in response to environmental changes, such as hypoxia. To identify distinct features of FLDM and BMDM response to hypoxia at the level of translation, we employed translating ribosome affinity purification (TRAP) to isolate polysomal RNA. RNA-seq profiling of translated RNA identified distinct hypoxia-induced translational signature of BMDM (Ly6e, vimentin and glycolysis-associated enzymes Pgk1, Tpi1, Aldoa, Ldha) and FLDM (chemokines Ccl7 and Ccl2). By translational profiling of BMDM and FLDM with deletion of the RNA-binding protein HuR, we identified transcripts that were dependent on HuR. These findings highlight the importance of HuR and identify its distinct targets for post-transcriptional regulation of gene expression in embryonic vs. adult-derived macrophages.

cell biology↗

bioRxiv2018

Virus-like vesicles expressing multiple antigens for immunotherapy of chronic hepatitis B

Infection with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, eventually progressing to liver fibrosis or cancer and causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B, relying on nucleoside antivirals and interferon, are inadequate and leave an unmet need for immunotherapeutic approaches. This report describes virus-like vesicles (VLV), a form of self-amplifying RNA replicons, which express multiple HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV). The HBV-VLV induces HBV-specific T cell responses to all three HBV antigens. Immunization of naive mice with the multiantigen HBV-VLV renders them resistant to acute challenge with HBV delivered by adeno-associated virus (AAV). Using a chronic model of HBV infection by AAV delivery of HBV, we demonstrate immunotherapeutic potential of the multiantigen HBV-VLV in combination with DNA booster immunization, as 40% of the HBV-VLV-treated mice showed a decline of the serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of multiantigen HBV-VLV for immunotherapy of chronic hepatitis B. IMPORTANCEMore than 240 million people worldwide are chronically infected with hepatitis B virus. Current therapies are not sufficiently effective and are often beyond reach in the developing world. We describe a virus-like vesicle-based immunotherapeutic vaccine that expresses three major antigens of hepatitis B virus as a self-amplifying RNA replicon. By incorporating three HBV antigens in a single vaccine, we ensure broad T cell responses. We demonstrate that immunization with this vaccine protects mice from hepatitis B virus in a model of acute challenge. Importantly, treatment with this vaccine shows 40% efficacy in a mouse model of chronic hepatitis B. Thus, this study paves the way for evaluation of the multi-antigen virus-like vesicles as a tool for immunotherapy of chronic hepatitis B.

immunology↗