Inhibition Of Target Gene Expression Research Articles

Transcriptome-Wide Evaluation Characterization of microRNAs and Assessment of Their Functional Roles as Regulators of Diapause in Ostrinia furnacalis Larvae (Lepidoptera: Crambidae).

microRNAs (miRNAs) function as vital regulators of diapause in insects through their ability to post-transcriptionally suppress target gene expression. In this study, the miRNA of Ostrinia furnacalis, an economically important global crop pest species, was characterized. For the included analyses, 9 small RNA libraries were constructed using O. furnacalis larvae in different diapause states (non-diapause, ND; diapause, D; diapause-termination, DT). The results identified 583 total miRNAs, of which 256 had previously been identified, whereas 327 were novel. Furthermore, comparison analysis revealed that 119 and 27 miRNAs were differentially expressed in the D vs. ND and DT vs. D, respectively. Moreover, the expression patterns of their miRNAs were also analyzed. GO and KEGG analysis of the target genes of differentially expressed miRNAs highlighted the importance of these miRNAs as diapause regulators in O. furnacalis, especially through metabolic processes, endocrine processes, 20-hydroxyecdysone, and circadian clock signaling pathways. In summary, this study highlighted the involvement of specific miRNAs in the control of diapause in O. furnacalis. To the best of our knowledge, this is the first study to identify miRNA expression patterns in O. furnacalis, thereby providing reference and novel evidence enhancing our current understanding of how small RNAs influence insect diapause.

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

Actinidia arguta Extract Containing Myo-Inositol Suppresses TNF-α-Induced VCAM-1 Expression and Monocyte Adhesion to Endothelial Cells via Inhibition of the PTEN/Akt/GSK-3β and NF-κB Signaling Pathways.

The primary inflammatory process in atherosclerosis, a major contributor to cardiovascular disease, begins with monocyte adhering to vascular endothelial cells. Actinidia arguta (kiwiberry) is an edible fruit that contains various bioactive components. While A. arguta extract (AAE) has been recognized for its anti-inflammatory characteristics, its specific inhibitory effect on early atherogenic events has not been clarified. We used tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs) for an in vitro model. AAE effectively hindered the attachment of THP-1 monocytes and reduced the expression of vascular cell adhesion molecule-1 (VCAM-1) in HUVECs. Transcriptome analysis revealed that AAE treatment upregulated phosphatase and tensin homolog (PTEN), subsequently inhibiting phosphorylation of AKT and glycogen synthase kinase 3β (GSK3β) in HUVECs. AAE further hindered phosphorylation of AKT downstream of the nuclear factor kappa B (NF-κB) signaling pathway, leading to suppression of target gene expression. Oral administration of AAE suppressed TNF-α-stimulated VCAM-1 expression, monocyte-derived macrophage infiltration, and proinflammatory cytokine expression in C57BL/6 mouse aortas. Myo-inositol, identified as the major compound in AAE, played a key role in suppressing THP-1 monocyte adhesion in HUVECs. These findings suggest that AAE could serve as a nutraceutical for preventing atherosclerosis by inhibiting its initial pathogenesis.

Expanding Roles of the E2F-RB-p53 Pathway in Tumor Suppression.

The transcription factor E2F links the RB pathway to the p53 pathway upon loss of function of pRB, thereby playing a pivotal role in the suppression of tumorigenesis. E2F fulfills a major role in cell proliferation by controlling a variety of growth-associated genes. The activity of E2F is controlled by the tumor suppressor pRB, which binds to E2F and actively suppresses target gene expression, thereby restraining cell proliferation. Signaling pathways originating from growth stimulative and growth suppressive signals converge on pRB (the RB pathway) to regulate E2F activity. In most cancers, the function of pRB is compromised by oncogenic mutations, and E2F activity is enhanced, thereby facilitating cell proliferation to promote tumorigenesis. Upon such events, E2F activates the Arf tumor suppressor gene, leading to activation of the tumor suppressor p53 to protect cells from tumorigenesis. ARF inactivates MDM2, which facilitates degradation of p53 through proteasome by ubiquitination (the p53 pathway). P53 suppresses tumorigenesis by inducing cellular senescence or apoptosis. Hence, in almost all cancers, the p53 pathway is also disabled. Here we will introduce the canonical functions of the RB-E2F-p53 pathway first and then the non-classical functions of each component, which may be relevant to cancer biology.

Cooperation of MLL1 and Jun in controlling H3K4me3 on enhancers in colorectal cancer

BackgroundEnhancer dysregulation is one of the important features for cancer cells. Enhancers enriched with H3K4me3 have been implicated to play important roles in cancer. However, their detailed features and regulatory mechanisms have not been well characterized.ResultsHere, we profile the landscape of H3K4me3-enriched enhancers (m3Es) in 43 pairs of colorectal cancer (CRC) samples. M3Es are widely distributed in CRC and averagely possess around 10% of total active enhancers. We identify 1322 gain variant m3Es and 367 lost variant m3Es in CRC. The target genes of the gain m3Es are enriched in immune response pathways. We experimentally prove that repression of CBX8 and RPS6KA5 m3Es inhibits target gene expression in CRC. Furthermore, we find histone methyltransferase MLL1 is responsible for depositing H3K4me3 on the identified Vm3Es. We demonstrate that the transcription factor AP1/JUN interacts with MLL1 and regulates m3E activity. Application of a small chemical inhibitor for MLL1 activity, OICR-9429, represses target gene expression of the identified Vm3Es, enhances anti-tumor immunity and inhibits CRC growth in an animal model.ConclusionsTaken together, our study illustrates the genome-wide landscape and the regulatory mechanisms of m3Es in CRC, and reveals potential novel strategies for cancer treatment.

RNA interference‐mediated silencing of NOA and V‐ATPase subunit D genes results in deformities and mortality in Bactrocera dorsalis (Hendel, 1912)

AbstractThe Oriental fruit fly, Bactrocera dorsalis (Hendel) is one of the five disastrous pests in the world with an economical significance. The guava germplasm resilient to fruit fly is not reported worldwide to date. The utilization of RNA interference (RNAi) approach can offer a promising alternative for fruit fly management in guava. The goal of our investigation is to evaluate the efficiency of dsRNA expression based on Escherichia coli used in B. dorsalis management by addressing its two indispensable genes, fatty acid elongase (NOA) and Vacuolar‐type H+‐ATPases D (v‐ATPase D). An artificial diet having E. coli strain HT115 (DE3) containing BdNOA, Bdv‐ATPase D dsRNA fed to maggots resulted in deformities with drastic mortality in treated adults, emerged pupae and maggots. The deformity together with total mortality in B. dorsalis was recorded at maximum as 74.66% and 64.20% in bacteria expressing dsRNAs of BdNOA and Bdv‐ATPase D, respectively, at 700 μL as compared to 350, 200 μL treatments. Severe deformities were reported such as underdeveloped abdomen, absence of wings, legs and inadequate eclosion along with suppressed target gene expression in B. dorsalis. This investigation thus provides a potential RNAi‐based pest management by targeting two key genes through feeding dsRNA‐expressing bacteria in B. dorsalis.

OC 23.3 Naked miRNA Mimics and siRNAs Selectively Transfect Megakaryocytes and Platelets In Vivo and Durably Suppress Target Gene Expression to Modulate Megakaryocyte and Platelet Function in Mice

OC 23.3 Naked miRNA Mimics and siRNAs Selectively Transfect Megakaryocytes and Platelets In Vivo and Durably Suppress Target Gene Expression to Modulate Megakaryocyte and Platelet Function in Mice

TgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9

CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14–15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.

Evaluation of the effectiveness of RNA silencing in the filamentous fungus Aspergillus niger using a binary vector integrated with Gateway recombination cloning technology

The filamentous fungus Aspergillus niger is widely employed in the production of citric acid and some industrial enzymes such as phytase, glucoamylase, and glucose oxidase. Currently, several genetic modification tools have been developed and applied to this fungal species. However, the RNA silencing technique is less used for A. niger. In the present study, an RNA silencing binary vector integrated with the Gateway recombination cloning technology was evaluated in A. niger. The integration of Gateway recombination cloning technology in binary vectors for rapid generation of hairpin RNA structure allowed the assessment of inhibition of target gene expression through mRNA degradation. The RNA silencing constructs were successfully transferred into A. nigervia Agrobacterium tumefaciens. Results showed that the RNA silencing vectors were effective in downregulating the expression of the DsRed fluorescent reporter gene and the stuA regulatory gene. Interestingly, the silenced mutants of stuA exhibited a significant reduction in fungal sporulation. This finding revealed that the RNA silencing technique with the examined Gateway binary vector represents a potential genetic tool for functional studies of target genes in A. niger.

Cationic Polymers as Transfection Reagents for Nucleic Acid Delivery.

Nucleic acid therapy can achieve lasting and even curative effects through gene augmentation, gene suppression, and genome editing. However, it is difficult for naked nucleic acid molecules to enter cells. As a result, the key to nucleic acid therapy is the introduction of nucleic acid molecules into cells. Cationic polymers are non-viral nucleic acid delivery systems with positively charged groups on their molecules that concentrate nucleic acid molecules to form nanoparticles, which help nucleic acids cross barriers to express proteins in cells or inhibit target gene expression. Cationic polymers are easy to synthesize, modify, and structurally control, making them a promising class of nucleic acid delivery systems. In this manuscript, we describe several representative cationic polymers, especially biodegradable cationic polymers, and provide an outlook on cationic polymers as nucleic acid delivery vehicles.

The role of Smo-Shh/Gli signaling activation in the prevention of neurological and ageing disorders.

Sonic hedgehog (Shh) signaling is an essential central nervous system (CNS) pathway involved during embryonic development and later life stages. Further, it regulates cell division, cellular differentiation, and neuronal integrity. During CNS development, Smo-Shh signaling is significant in the proliferation of neuronal cells such as oligodendrocytes and glial cells. The initiation of the downstream signalling cascade through the 7-transmembrane protein Smoothened (Smo) promotes neuroprotection and restoration during neurological disorders. The dysregulation of Smo-Shh is linked to the proteolytic cleavage of GLI (glioma-associated homolog) into GLI3 (repressor), which suppresses target gene expression, leading to the disruption of cell growth processes. Smo-Shh aberrant signalling is responsible for several neurological complications contributing to physiological alterations like increased oxidative stress, neuronal excitotoxicity, neuroinflammation, and apoptosis. Moreover, activating Shh receptors in the brain promotes axonal elongation and increases neurotransmitters released from presynaptic terminals, thereby exerting neurogenesis, anti-oxidation, anti-inflammatory, and autophagy responses. Smo-Shh activators have been shown in preclinical and clinical studies to help prevent various neurodegenerative and neuropsychiatric disorders. Redox signalling has been found to play a critical role in regulating the activity of the Smo-Shh pathway and influencing downstream signalling events. In the current study ROS, a signalling molecule, was also essential in modulating the SMO-SHH gli signaling pathway in neurodegeneration. As a result of this investigation, dysregulation of the pathway contributes to the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).Thus, Smo-Shh signalling activators could be a potential therapeutic intervention to treat neurocomplications of brain disorders.

Abstract LB133: An orally bioavailable degrader targeting androgen receptor and the splice variant in castration resistant prostate cancer

Abstract The development and progression of prostate cancer depends on androgen receptor (AR) whose sustained activity albeit the use of anti-androgen drugs accounts for one of the major mechanisms leading to drug resistance. As such, the clinical benefits of current therapeutic agents remain rather limited for the advanced prostate cancer. Given that expression of constitutively active AR variants (AR-Vs) that lack the ligand-binding domain (LBD) clinically associates with the drug resistance and poor survival, targeting both AR and the LBD-truncated AR-Vs is anticipated an effective approach to overcome therapy resistance. Here, we present Proteolysis Targeting Chimeras (PROTAC) compound ITRI-148, capable of effectively degrading both full-length and LBD-truncated AR by targeting the proteins via the N-terminal domain to Cereblon-mediated ubiquitination and degradation. Degradation of AR and AR-V7 suppresses target gene expression, and inhibits cell proliferation with accompanied apoptosis activation in AR-V7-expressing castration-resistant prostate cancer (CRPC) cell models. In vivo, this compound displays good pharmacokinetics profiles and oral bioavailability with strong antitumor efficacy towards castration-, enzalutamide-resistant CWR22Rv1 and VCaP xenograft models. These data provide compelling evidence that by targeting AR NTD for induced degradation of active AR protein variants, ITRI-148 is a promising agent for the intervention of advanced therapy-resistant prostate cancers. Citation Format: Chiu-Lien Hung, Ling-Yu Wang, Chih-Wei Fu, Hung-Chih Hsu, Hsin-Jien Kung. An orally bioavailable degrader targeting androgen receptor and the splice variant in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB133.

RNA INTERFERENCE IN THRIPS VECTORS: A STEP FORWARD TOWARD SUSTAINABLE MANAGEMENT

Thrips are major pests of agricultural and horticultural crops worldwide. In addition to causing direct damage, thrips are the most common vector of tospoviruses. Currently, thrips control is mostly based on chemical insecticides, but effective control can be difficult to achieve because it has developed resistance to various insecticides. Therefore, there is an urgent need for novel alternative management options. A viable strategy for the management of the virus-vector complex involves understanding the molecular links between thrips and viruses and disrupting their association. RNA interference, a sequence-specific method to suppress target gene expression, is a conserved process in all eukaryotic organisms and has emerged as a new tool for reverse genetics and potential pest control. Advancements in RNA interference-based gene silencing techniques in the past few years could potentially be utilized in the management of the thrips-tospovirus complexes. RNA interference-mediated plant protection strategies have the potential to revolutionize pest management practices in a sustainable and effective way if development is carried out profoundly by discerning bioinformatics identification, in-silico designing of RNAi trigger molecules, laboratory and field-based toxicity and biosafety investigations.

Unravel the regulatory mechanism of Yrr1p phosphorylation in response to vanillin stress in Saccharomyces cerevisiae

Improving the resistance of Saccharomyces cerevisiae to vanillin, derived from lignin, will benefit the design of robust cell factories for lignocellulosic biorefining. The transcription factor Yrr1p mediates S. cerevisiae resistance to various compounds. In this study, eleven predicted phosphorylation sites were mutated, among which 4 mutants of Yrr1p, Y134A/E and T185A/E could improve vanillin resistance. Both dephosphorylated and phosphorylated mutations at Yrr1p 134 and 185 gathered in the nucleus regardless of the presence or absence of vanillin. However, the phosphorylated mutant Yrr1p inhibited target gene expression, while dephosphorylated mutants promoted expression. Transcriptomic analysis showed that the dephosphorylated Yrr1p T185 mutant, under vanillin stress, upregulated ribosome biogenesis and rRNA processing. These results demonstrate the mechanism by which Yrr1p phosphorylation regulates the expression of target genes. The identification of key phosphorylation sites in Yrr1p offers novel targets for the rational construction of Yrr1p mutants to improve resistance to other compounds.

The Essential Function of miR-5739 in Embryonic Muscle Development.

Embryologically, mesodermal development is closely related to the development of various organs such as muscles, blood vessels, and hearts, which are the main organs that make up the body. However, treatment for mesoderm developmental disorders caused by congenital or acquired factors has so far relied on surgery and drug treatment for symptom relief, and more fundamentally, treatment for mesoderm developmental disorders is needed. In our study, microRNA (miRNA), which plays an important role in the mesoderm development process, was identified and the developmental function was evaluated. miRNAs consist of small nucleotides, which act as transcription factors that bind to the 3' untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of miR-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease. Our study demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regulate muscle and vascular differentiation during embryonic development.

Transplastomic tomatoes expressing double-stranded RNA against a conserved gene are efficiently protected from multiple spider mites.

Spider mites are serious pests and have evolved significant resistance to many chemical pesticides, thus making their control challenging. Several insect pests can be combated by plastid-mediated RNA interference (PM-RNAi), but whether PM-RNAi can be utilized to control noninsect pests is unknown. Here, we show that three species of spider mites (Tetranychus evansi, Tetranychus truncatus, and Tetranychus cinnabarinus) take up plastid RNA upon feeding. We generated transplastomic tomato plants expressing double-stranded RNA (dsRNA) targeted against a conserved region of the spider mite β-Actin mRNA. Transplastomic plants exhibited high levels of resistance to all three spider mite species, as evidenced by increased mortality and suppression of target gene expression. Notably, transplastomic plants induced a more robust RNAi response, caused higher mortality, and were overall better protected from spider mites than dsRNA-expressing nuclear transgenic plants. Our data demonstrate the potential of PM-RNAi as an efficient pest control measure for spider mites and extend the application range of the technology to noninsect pests.

Regulation of Normal and Neoplastic Proliferation and Metabolism by the Extended Myc Network.

The Myc Network, comprising a small assemblage of bHLH-ZIP transcription factors, regulates many hundreds to thousands of genes involved in proliferation, energy metabolism, translation and other activities. A structurally and functionally related set of factors known as the Mlx Network also supervises some of these same functions via the regulation of a more limited but overlapping transcriptional repertoire. Target gene co-regulation by these two Networks is the result of their sharing of three members that suppress target gene expression as well as by the ability of both Network's members to cross-bind one another's consensus DNA sites. The two Networks also differ in that the Mlx Network's control over transcription is positively regulated by several glycolytic pathway intermediates and other metabolites. These distinctive properties, functions and tissue expression patterns potentially allow for sensitive control of gene regulation in ways that are differentially responsive to environmental and metabolic cues while allowing for them to be both rapid and of limited duration. This review explores how such control might occur. It further discusses how the actual functional dependencies of the Myc and Mlx Networks rely upon cellular context and how they may differ between normal and neoplastic cells. Finally, consideration is given to how future studies may permit a more refined understanding of the functional interrelationships between the two Networks.

Roles of transducin-like enhancer of split (TLE) family proteins in tumorigenesis and immune regulation.

Mammalian transducin-like enhancer of split family proteins (TLEs) are homologous to Drosophila Groucho (Gro) and are essential transcriptional repressors. Seven TLE family members, TLE1-7, have been identified to date. These proteins do not bind DNA directly; instead, they bind a set of transcription factors and thereby inhibit target gene expression. Loss of TLEs in mice usually leads to defective early development; however, TLE functions in developmentally mature cells are unclear. Recent studies have revealed that TLEs are dysregulated in certain human cancer types and may function as oncogenes or tumor suppressors in different contexts. TLE levels also affect the efficacy of cancer treatments and the development of drug resistance. In addition, TLEs play critical roles in the development and function of immune cells, including macrophages and lymphocytes. In this review, we provide updates on the expression, function, and mechanism of TLEs; discuss the roles played by TLEs in tumorigenesis and the inflammatory response; and elaborate on several TLE-associated signaling pathways, including the Notch, Wnt, and MAPK pathways. Finally, we discuss potential strategies for targeting TLEs in cancer therapy.

Abstract 6304: Targeting HIF2a with siRNA: From preclinical models to the clinic

Abstract Hypoxia-inducible factor 2 alpha (HIF2a) is arguably the most important driver of kidney cancer. HIF2a is constitutively activated following von Hippel-Lindau (VHL) gene inactivation, the signature event of the most common type of kidney cancer, clear cell renal cell carcinoma (ccRCC). HIF2a functions as a heterodimeric transcription factor and regulates a program of gene expression that promotes cell proliferation, stemness, and angiogenesis. Using a highly specific inhibitor designed to target a structural vulnerability in HIF2a (PT2399), we previously showed that approximately 50% of ccRCCs are dependent on HIF2a. However, prolonged drug exposure results in resistance and the acquisition of gatekeeper mutations, which we reported first in patient-derived xenografts (PDXs) and subsequently in humans. Using the same PDX platform that previously validated PT2399, we show that HIF2a can be effectively inhibited using a tumor-directed siRNA (siHIF2). Referring herein to both first- and second-generation (ARO-HIF2) siRNA drugs, siHIF2 is specifically taken up by human ccRCC tumors transplanted in mice, where it depletes HIF2a inhibiting target gene expression and tumor growth. Through orthogonal RNA-seq studies integrating both PT2399 and siHIF2 in PDXs, we provide unprecedented detail on the HIF2a effector transcriptome, which we further dissect by incorporating ChIP-seq. A PDX line was generated from a ccRCC patient who had paraneoplastic polycythemia (a HIF2a dependent syndrome due to erythropoietin [Epo] secretion by the tumor) and participated in the phase I trial of ARO-HIF2 (NCT04169711). We show that siHIF2 effectively depleted HIF2a in both the PDX as well as in the patient, that it normalized Epo (and hemoglobin), and that it inhibited tumor growth. siHIF2 has activity against both wild-type and drug-resistant mutant HIF2a and is expected to be active in patients progressing on PT2977 (belzutifan), a PT2399-related drug recently approved by the FDA. To our knowledge, this is the first example of functional inactivation of an oncoprotein with a tumor-directed siRNA in humans. In summary, these studies provide unique insight into HIF2a (the only known core dependency in ccRCC), illustrate how it can be effectively inhibited by an siRNA drug, and establish a paradigm for the development of tumor directed siRNA-based therapeutics. Citation Format: Yuanqing Ma, Christina Stevens, Olivia Brandenburg, Vanina Toffessi Tcheuyap, Quyen N. Do, Faeze Saatchi, Tanner Hardy, Oluwatomilade Fatunde, Alyssa Macchiaroli, Jeffrey Miyata, Deyssy Carrillo, Thomas Schluep, So Wong, Alana Christie, Payal Kapur, Ivan Pedrosa, James Hamilton, James Brugarolas. Targeting HIF2a with siRNA: From preclinical models to the clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6304.

Aerosol delivery of star polymer-siRNA nanoparticles as a therapeutic strategy to inhibit lung tumor growth

Lung cancer is a major contributor to cancer-related death worldwide. siRNA nanomedicines are powerful tools for cancer therapeutics. However, there are challenges to overcome to increase siRNA delivery to solid tumors, including penetration of nanoparticles into a complex microenvironment following systemic delivery while avoiding rapid clearance by the reticuloendothelial system, and limited siRNA release from endosomes once inside the cell. Here we characterized cell uptake, intracellular trafficking, and gene silencing activity of miktoarm star polymer (PDMAEMA-POEGMA) nanoparticles (star nanoparticles) complexed to siRNA in lung cancer cells. We investigated the potential of nebulized star-siRNA nanoparticles to accumulate into orthotopic mouse lung tumors to inhibit expression of two genes [βIII-tubulin, Polo-Like Kinase 1 (PLK1)] which: 1) are upregulated in lung cancer cells; 2) promote tumor growth; and 3) are difficult to inhibit using chemical drugs. Star-siRNA nanoparticles internalized into lung cancer cells and escaped the endo-lysosomal pathway to inhibit target gene expression in lung cancer cells in vitro. Nebulized star-siRNA nanoparticles accumulated into lungs and silenced the expression of βIII-tubulin and PLK1 in mouse lung tumors, delaying aggressive tumor growth. These results demonstrate a proof-of-concept for aerosol delivery of star-siRNA nanoparticles as a novel therapeutic strategy to inhibit lung tumor growth.