Reduced Target Gene Expression Research Articles

HIGH PLOIDY2-mediated SUMOylation of transcription factor ARR1 controls two-component signaling in Arabidopsis.

Cytokinins regulate plant growth, development, and responses to environmental stresses such as cold via phosphorelay from cytokinin receptors to the ARABIDOPSIS RESPONSE REGULATORs (ARRs). However, the molecular mechanisms underlying the activation of type-B ARR transcriptional activity in Arabidopsis (Arabidopsis thaliana) remain unclear. Here, we show that the E3 SUMO ligase HIGH PLOIDY2 SUMOylates ARR1, a type-B ARR, at K236, triggering its activation. Cold- or cytokinin-induced phosphorylation of ARR1 at D89 is crucial for its interaction with HPY2. Lysine 236 is critical for ARR1's transactivation without compromising its DNA-binding ability, while D89 is crucial for ARR1's binding to target gene promoters. Cytokinin enhances ARR1's chromatin binding, but cold does not. ARR1 K236 plays a critical role in promoting histone H3 acetylation in response to both cytokinin and cold without affecting chromatin binding. The K236R mutation in ARR1 reduces target gene expression and alters cytokinin and cold response phenotypes. This study unveils a mechanism of ARR1 activation wherein phosphorylated ARR1 interacts with HPY2 and binds to chromatin in response to cytokinin. Cold triggers a phosphorelay targeting chromatin-bound ARR1. HPY2 then catalyzes ARR1 SUMOylation at K236, enhancing histone H3 acetylation and leading to transcriptional activation of ARR1 in response to both cold and cytokinin.

Oral Administration of PLGA Nanoparticles to Deliver Antisense Oligonucleotides to Inflammatory Lesions in the Gastrointestinal Tract.

In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of inflammatory bowel disease (IBD). We chose a water-in-oil-in-water (w/o/w) method to prepare the NPs using poly(lactide-co-glycolide) as a matrix and Pluronic as a stabilizer. The obtained NPs had a suitable diameter (158 nm) for the penetration of the mucus layer, endocytic uptake by enterocytes, and accumulation in inflammatory lesions in the intestine. The amount of ASOs in the NPs was relatively large (6.41% (w/w)). When the NPs were stably dispersed in solutions that mimicked gastrointestinal (GI) juice, minimal leakage of ASOs was demonstrated over the required period. The NPs were administered orally to mice with colitis induced by dextran sodium sulfate, which reduced target gene expression in the colons and rectums of the mice, whereas naked ASO administration caused no reduction in gene expression. Thus, the NPs have the potential of promising oral carriers of ASOs for the treatment of IBD that specifically target inflammatory lesions in the GI tract, thereby reducing the non-specific toxic effects of ASOs.

Generation of DNAzyme in Bacterial Cells by a Bacterial Retron System.

DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10-23 (Dz 10-23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequence-specific mRNA cleavage. However, the in vivo application of Dz 10-23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10-23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10-23 by cloning the Dz 10-23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cfp encoding cyan fluorescence protein and other functional genes such as mreB and ftsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10-23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10-23 but also demonstrated that Dz 10-23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.

Non-Toxicological Role of Aryl Hydrocarbon Receptor in Obesity-Associated Multiple Myeloma Cell Growth and Survival.

Obesity is not only a risk factor for multiple myeloma (MM) incidence, but it is also associated with an increased risk of progression from myeloma precursors-monoclonal gammopathy of undetermined significance-and smoldering myeloma. Adipocytes in the bone marrow (BMAs) microenvironment have been shown to facilitate MM cell growth via secreted factors, but the nature of these secreted factors and their mechanism of action have not been fully elucidated. The elevated expression of aryl hydrocarbon receptor (AhR) is associated with a variety of different cancers, including MM; however, the role of AhR activity in obesity-associated MM cell growth and survival has not been explored. Indeed, this is of particular interest as it has been recently shown that bone marrow adipocytes are a source of endogenous AhR ligands. Using multiple in vitro models of tumor-adipocyte crosstalk to mimic the bone microenvironment, we identified a novel, non-toxicological role of the adipocyte-secreted factors in the suppression of AhR activity in MM cells. A panel of six MM cell lines were cultured in the presence of bone marrow adipocytes in (1) a direct co-culture, (2) a transwell co-culture, or (3) an adipocyte-conditioned media to interrogate the effects of the secreted factors on MM cell AhR activity. Nuclear localization and the transcriptional activity of the AhR, as measured by CYP1A1 and CYP1B1 gene induction, were suppressed by exposure to BMA-derived factors. Additionally, decreased AhR target gene expression was associated with worse clinical outcomes. The knockdown of AhR resulted in reduced CYP1B1 expression and increased cellular growth. This tumor-suppressing role of CYP1A1 and CYP1B1 was supported by patient data which demonstrated an association between reduced target gene expression and worse overall survival. These data demonstrated a novel mechanism by which bone marrow adipocytes promote MM progression.

Design of siRNA Bioconjugates for Efficient Control of Cancer-Associated Membrane Receptors.

Research on siRNA delivery has seen tremendous growth over the past few decades. As one of the major delivery strategies, siRNA bioconjugates offer the potential to enhance and extend the pharmacological properties of siRNAs while minimizing toxicity. In this paper, we suggest the development of a siRNA conjugate platform with peptides and proteins that are ligands of target receptors for cancer treatment. The siRNA bioconjugates target and block the receptor membrane proteins, enter the cells through receptor-mediated endocytosis, and inhibit the expression of that same target membrane receptor, thereby doubly controlling the function of the membrane proteins. The three kinds of bioconjugates targeting CD47, PD-L1, and EGFR were synthesized via two different copper-free click chemistry reactions. Results showed the cellular uptake of each conjugate, reduction of target gene expression, and efficient functional control of receptor proteins. This platform provides an effective approach for regulating membrane proteins in various diseases beyond cancer.

Abstract 3721: The C1 domain of capicua has functional importance in human CIC-DUX4

Abstract Genomic rearrangements between the HMG-box family transcriptional repressor capicua (CIC) and multiple partner genes result in fusion oncoproteins that activate transcription to drive sarcoma growth and progression. In the case of CIC-DUX4, the most common CIC-rearranged fusion oncoprotein, the fusion often retains over 90% of the wild type CIC protein structure including the HMG box and C-terminal C1 domain. Structure-function studies in Drosophila have suggested that the C1 domain of CIC, a non-HMG box DNA binding domain, is important for the transactivating capacity of a synthetic fly-human CIC-DUX4. Consistent with these findings, we performed a retrospective analysis of over 70 breakpoints described from CIC-DUX4 human derived tumors, which revealed that the C1 domain is structurally conserved within the context of the CIC-DUX4 fusion. To further understand the functional role of the C1 domain in human CIC-DUX4 we generated human CIC-DUX4 C1 domain and HMG-box deletion (alone or in combination) mutant constructs and performed a series of studies to test the functional impact on CIC-DUX4 protein levels, subcellular localization, and transcriptional activity. Through these analyses we have found that deletion of the C1 domain does not impact CIC-DUX4 oncoprotein expression or localization but does dramatically reduce target gene expression. In ongoing in vivo studies, we aim to evaluate the significance of the C1 domain in inducing CIC-DUX4 driven tumorigenesis. Collectively, our findings validate the functional importance of the C1 domain in human CIC-DUX4 and to our knowledge provide the first harmonized analysis of breakpoint locations in CIC-rearranged sarcoma. Citation Format: Cuyler Luck, Kyle A. Jacobs, Ross A. Okimoto. The C1 domain of capicua has functional importance in human CIC-DUX4. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3721.

Analysis of MicroRNA Regulation and Gene Expression Variability in Single Cell Data.

MicroRNAs (miRNAs) regulate gene expression by binding to mRNAs, and thus reduce target gene expression levels and expression variability, also known as ‘noise’. Single-cell RNA sequencing (scRNA-seq) technology has been used to study miRNA and mRNA expression in single cells. To evaluate scRNA-seq as a tool for investigating miRNA regulation, we analyzed datasets with both mRNA and miRNA expression in single-cell format. We found that miRNAs slightly reduce the expression noise of target genes; however, this effect is easily masked by strong technical noise from scRNA-seq. We suggest improvements aimed at reducing technical noise, which can be implemented in experimental design and computational analysis prior to running scRNA-seq. Our study provides useful guidelines for experiments that evaluate the effect of miRNAs on mRNA expression from scRNA-seq.

A Comparison of RNA Interference via Injection and Feeding in Honey Bees.

Simple SummaryRNA interference is an important way to analyze gene function. It is also widely used in honey bees. It was thought that RNA interference with honey bee brain genes could only be achieved by injection. However, this method of injection is very complicated and can easily cause damage to bees. Recent studies have shown that specially treated siRNA can knockdown brain genes in animals by feeding them. Therefore, we used different approaches to deliver modified and unmodified siRNAs to investigate what methods can successfully interfere with honey bee brain genes. The results of this study are helpful to improve the application of RNA interference techniques in honey bees and other insects.RNA interference (RNAi) has been used successfully to reduce target gene expression and induce specific phenotypes in several species. It has proved useful as a tool to investigate gene function and has the potential to manage pest populations and reduce disease pathogens. However, it is not known whether different administration methods are equally effective at interfering with genes in bees. Therefore, we compared the effects of feeding and injection of small interfering RNA (siRNA) on the messenger RNA (mRNA) levels of alpha-aminoadipic semialdehyde dehydrogenase (ALDH7A1), 4-coumarate-CoA ligase (4CL), and heat shock protein 70 (HSP70). Both feeding and injection of siRNA successfully knocked down the gene but feeding required more siRNA than the injection. Our results suggest that both feeding and injection of siRNA effectively interfere with brain genes in bees. The appropriateness of each method would depend on the situation.

Localized efficacy of environmental RNAi in Tetranychus urticae

Environmental RNAi has been developed as a tool for reverse genetics studies and is an emerging pest control strategy. The ability of environmental RNAi to efficiently down-regulate the expression of endogenous gene targets assumes efficient uptake of dsRNA and its processing. In addition, its efficiency can be augmented by the systemic spread of RNAi signals. Environmental RNAi is now a well-established tool for the manipulation of gene expression in the chelicerate acari, including the two-spotted spider mite, Tetranychus urticae. Here, we focused on eight single and ubiquitously-expressed genes encoding proteins with essential cellular functions. Application of dsRNAs that specifically target these genes led to whole mite body phenotypes—dark or spotless. These phenotypes were associated with a significant reduction of target gene expression, ranging from 20 to 50%, when assessed at the whole mite level. Histological analysis of mites treated with orally-delivered dsRNAs was used to investigate the spatial range of the effectiveness of environmental RNAi. Although macroscopic changes led to two groups of body phenotypes, silencing of target genes was associated with the distinct cellular phenotypes. We show that regardless of the target gene tested, cells that displayed histological changes were those that are in direct contact with the dsRNA-containing gut lumen, suggesting that the greatest efficiency of the orally-delivered dsRNAs is localized to gut tissues in T. urticae.

Dissection of the microRNA Network Regulating Hedgehog Signaling in Drosophila.

The evolutionarily conserved Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. Aberrant Hh signaling often leads to various forms of developmental anomalies and cancer. Since altered microRNA (miRNA) expression is associated with developmental defects and tumorigenesis, it is not surprising that several miRNAs have been found to regulate Hh signaling. However, these miRNAs are mainly identified through small-scale in vivo screening or in vitro assays. As miRNAs preferentially reduce target gene expression via the 3′ untranslated region, we analyzed the effect of reduced expression of core components of the Hh signaling cascade on downstream signaling activity, and generated a transgenic Drosophila toolbox of in vivo miRNA sensors for core components of Hh signaling, including hh, patched (ptc), smoothened (smo), costal 2 (cos2), fused (fu), Suppressor of fused (Su(fu)), and cubitus interruptus (ci). With these tools in hand, we performed a genome-wide in vivo miRNA overexpression screen in the developing Drosophila wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the in vivo Hh regulatory network. Moreover, these miRNAs may act as general regulators of Hh signaling, as their overexpression disrupts Hh signaling-mediated cyst stem cell maintenance during spermatogenesis. To identify direct targets of these newly discovered miRNAs, we used the miRNA sensor toolbox to show that miR-10 and miR-958 directly target fu and smo, respectively, while the other five miRNAs act through yet-to-be-identified targets other than the seven core components of Hh signaling described above. Importantly, through loss-of-function analysis, we found that endogenous miR-10 and miR-958 target fu and smo, respectively, whereas deletion of the other five miRNAs leads to altered expression of Hh signaling components, suggesting that these seven newly discovered miRNAs regulate Hh signaling in vivo. Given the powerful effects of these miRNAs on Hh signaling, we believe that identifying their bona fide targets of the other five miRNAs will help reveal important new players in the Hh regulatory network.

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes.

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference (RNAi) Administration to Adult <em>Anopheles gambiae</em> Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Exogenous administration of dsRNA for the demonstration of RNAi in Maruca vitrata (lepidoptera: crambidae).

The online version contains supplementary material available at 10.1007/s13205-021-02741-8.

Multi-modal effects of 1B3, a novel synthetic miR-193a-3p mimic, support strong potential for therapeutic intervention in oncology.

Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer.

Analysis of microRNA Regulation in Single Cells.

MicroRNAs (miRNAs) regulate gene expression by binding to mRNAs. Consequently, they reduce target gene expression levels and expression variability, also known as "noise." Single-cell RNA sequencing (scRNA-seq) technology has been used to study miRNA and mRNA expression in single cells, and has demonstrated its strength in quantifying cell-to-cell variation. Here we describe how to investigate miRNA regulation using data with both mRNA and miRNA expression in single cell format. We show that miRNAs reduce the expression levels and also expression noise of target genes in single cells. Finally, we also discuss potential improvements in experimental design and computational analysis of scRNA-seq in order to reduce or partition the technical noise.

Primary effusion lymphoma enhancer connectome links super-enhancers to dependency factors

Primary effusion lymphoma (PEL) has a very poor prognosis. To evaluate the contributions of enhancers/promoters interactions to PEL cell growth and survival, here we produce H3K27ac HiChIP datasets in PEL cells. This allows us to generate the PEL enhancer connectome, which links enhancers and promoters in PEL genome-wide. We identify more than 8000 genomic interactions in each PEL cell line. By incorporating HiChIP data with H3K27ac ChIP-seq data, we identify interactions between enhancers/enhancers, enhancers/promoters, and promoters/promoters. HiChIP further links PEL super-enhancers to PEL dependency factors MYC, IRF4, MCL1, CCND2, MDM2, and CFLAR. CRISPR knock out of MEF2C and IRF4 significantly reduces MYC and IRF4 super-enhancer H3K27ac signal. Knock out also reduces MYC and IRF4 expression. CRISPRi perturbation of these super-enhancers by tethering transcription repressors to enhancers significantly reduces target gene expression and reduces PEL cell growth. These data provide insights into PEL molecular pathogenesis.

Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides

The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.

MicroRNAs and Uveal Melanoma: Understanding the Diverse Role of These Small Molecular Regulators

Uveal melanoma (UM) is a rare tumour of the eye, characterised by a high propensity to metastasise in half of all patients, most frequently to the liver. Although there are effective treatment options for the primary tumour, once metastasis has occurred prognosis is poor, with overall survival limited to months. Currently, there are no effective treatments for metastatic UM, despite the tumour having a well-defined signalling pathway to which many therapies have been directed. In an effort to develop novel treatment approaches, understanding the role of other signalling molecules, such as microRNAs, is fundamental. MicroRNAs (miRNAs) are small non-coding RNA molecules involved in posttranscriptional gene regulation, resulting in reduced target gene expression and subsequent protein translation. In UM, several dysregulated miRNAs have been proposed to play a functional role in disease progression, whereas others have been put forward as clinical biomarkers of high-risk disease following isolation from blood, plasma and exosomes. Most recently, analyses of large datasets have identified promising prognostic miRNA signatures and panels. This review navigates the plethora of aberrant miRNAs disclosed so far in UM, and maps these to signalling pathways, which could be targeted in future therapies for the disseminated disease.

Host-induced gene silencing of multiple genes of Fusarium graminearum enhances resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB) caused primarily by Fusarium graminearum is a devastating disease of wheat worldwide. FHB infection not only leads to serious yield lossses, but also results in critical health issues because of mycotoxin contamination, especially deoxynivalenol (DON). Novel approaches are therefore urgently required to combat FHB, due to the development of fungicide resistance in F. graminearum and the unavailability of FHB-resistant wheat cultivars. We here evaluated the use of host-induced gene silencing (HIGS) in transgenic wheat plants as a strategy to control FHB by simultaneously silencing multiple genes of F. graminearum. A chimeric hairpin RNA interference (RNAi) construct targeting to the pathogenicity and essential genes including FgSGE1, FgSTE12, and FgPP1 was able to simultaneously reduce target gene expression and caused defects of growth, conidiation and virulence in transgenic F. graminearum strains. Two transgenic wheat lines of T2 and T3 generations expressing the RNAi construct in a susceptible wheat cultivar Yangmai16 displayed high levels of stable and consistent resistance to F. graminearum, and reduced DON production in wheat. Development of fungal infection structures was dramatically impaired in the transgenic wheat lines. Small RNA sequencing confirmed the presence of small interfering RNAs specific to the three targeted fungal genes in the transgenic wheat plants, which is consistent with the reduced expression of the target genes in the invading fungus. These results demonstrate that HIGS targeting multiple genes of the fungus is effective and can be used as an alternative approach for developing FHB-resistant crops.

Abstract B057: Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown

Abstract Triple-negative (TNBC) and HER2+ breast cancers (BCs) are especially aggressive tumors, which are prone to relapse and metastasize post chemo- or targeted therapy. Immunotherapy provides a promising, but unproven, alternative approach for treating poor-prognosis BCs. BCs have relatively low nonsynonymous mutation rates, suggesting that many BCs will respond poorly to immunotherapy. Novel strategies to increase BC cell immunogenicity and improve tumor-antigen specific T-cell responses are needed to enhance the efficacy of BC immune therapy. We aim to enhance the immunogenicity of breast tumor by taking advantage of the unique strength of EpCAM-aptamer conjugated small interfering RNAs (AsiCs), which can knock down gene expression selectively in EpCAM+ BC cells, to make aggressive BCs visible to T-cells and improve T-cell tumor recruitment and function. EpCAM is a tumor-specific antigen expressed at several logs higher levels on all epithelial cancers and cancer stem cells relative to normal epithelia. The EpCAM aptamer our lab developed binds with high affinity to both mouse and human EpCAM. Using this platform, we investigated the use of EpCAM-AsiCs for BC immune modulation by targeting genes controlling different functional processes: 1) knocking down the nonsense-mediated mRNA decay (NMD) gene UPF2, to elicit tumor neoantigen expression to improve BC tumor recognition by the immune system; 2) knocking down CD47 to promote phagocytosis of cancer cells and enhance cross-presentation of tumor antigens; 3) knocking down DNA repair enzyme PARP1 to produce more DNA damage and consequent genetic mutations, thereby introducing tumor-specific neoantigens to the immune system; 4) knocking down PLK1 which is essential for cell mitosis, and MCL1, which is a critical survival factor in TNBC, to directly kill tumor cells. The increased tumor cell death induced by PLK1 and MCL1 knockdown could potentially promote tumor antigen cross-presentation to CD8+ T-cells. Our data indicate that each of these AsiCs potently silences gene expression within mouse or human BC cell lines in vitro, and efficiently reduce target gene expression in EpCAM+ tumor cells in vivo. When treating mice bearing 4T1E (4T1 cell line with high EpCAM expression) tumors, EpCAM-AsiCs targeting UPF2, CD47, PARP1, PLK1 and MCL1 each significantly suppressed 4T1E tumor growth, increased CD8+ T-cell tumor infiltration, CD8+ T-cell/regulatory T-cells (Treg) ratio, the effector functions and degranulation capacity of CD8+ TILs compared to those of the control group. The capacity of these CD8+ TILs to directly kill tumor cells was also improved. Tumor-associated macrophages (TAMs) from CD47-AsiC treated tumors showed improved phagocytic capacity as determined by in vitro and in vivo phagocytosis assays. CD47-AsiC also increased the ratio of M1 (tumor suppressive) to M2 (tumor promoting) TAMs. Dendritic cells (DCs) from CD47-AsiC treated tumors show enhanced CD40 and CD86 expression, suggesting an improved immunostimulatory capacity. Using antibody depletion assay, we further show that CD4+ T-cells, CD8+ T-cells and macrophages all contribute to the tumor inhibition capacity of CD47-AsiC; and TAMs/DC play an important role in stimulating the functions of CD8+ TILs. In addition, we show that CD47-AsiC outperforms the anti-CD47 antibody in suppressing 4T1E tumor progression. Finally, we show that UPF2, CD47, PARP1, PLK1 and MCL1 AsiCs work in synergy to further delay tumor growth, and even lead to tumor regression, compared to single AsiC treatment. The combination therapy significantly increased the amount of CD8+ TILs, reduced the Tregs and myeloid-derived suppressor cells (MDSCs) in the tumor, and improved the functions of both CD4+ and CD8+ TILs. Our data suggest that these novel EpCAM-AsiCs, with their small size and high selectivity, have great potential to improve therapeutic efficacy and reduce toxicity for BC patients compared to current checkpoint inhibitors. Citation Format: Ying Zhang, Judy Lieberman. Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B057.