Browsing by Subject "RNA, Small Interfering"
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Item Exploration of Nuclear Receptor Activity and siRNA-Derived Phenotypes as Therapeutics in Non-Small Cell Lung Cancer(2015-04-10) Carstens, Ryan Murray; Kittler, Ralf; Minna, John D.; Mangelsdorf, David J.; Kliewer, Steven A.; White, Michael A.Nuclear hormone receptors are master regulators of diverse cellular functions implicated in tumor pathogenesis and as oncogenic drivers of many human cancers. To better understand what role these important receptors might be playing in lung cancer, three interconnected studies were initiated to assess nuclear receptor function, expression, and drugability within the lung cancer context. First, a "technology development" project was undertaken to produce and troubleshoot a CLIA-certifiable, high-throughput biomarker platform capable of mRNA expression signature assessment from FFPE specimens. The platform was used to assess NR/CoReg expression levels across a 500+ sample FFPE dataset. Categorical NR/CoReg downregulation upon tumor progression as well as survival benefits for patients retaining a non-pathological NR/CoReg expression pattern were discovered. Second, a panel of 110 NR ligands was screened across a 100-member cell line panel representative of all clinically-relevant facets of lung and breast cancers to pharmacologically interrogate these receptors as novel lung cancer targets. Following completion of this screening effort, three classes of ligands targeting the estrogen, glucocorticoid, and vitamin D receptors (ER, GR, and VDR respectively) that exert anti-proliferative phenotypes on specific subsets of the lung cancer cell lines were identified. Of particular note, several of these agents are routinely used in current clinical practice (particularly dexamethasone) and represent excellent candidates for rapid clinical translation of these findings. Finally, an RNAi-based systematic functional interrogation of NR/CoReg function was undertaken in a 100+ member cell line panel representative of all clinically-relevant facets of lung and breast cancers. A reproducible classification of lung and breast cancers was defined based on their holistic functional states as represented by the RNAi dataset. Each of these "clades" of cancer cell lines was demonstrated to be specifically targetable by unique siRNA reagents capable of inducing growth attenuation or amplification in only that clade of cell lines. Further investigation into the mechanisms of action of these siRNA reagents unexpectedly revealed that the phenotypes were largely mediated by miRNA-like seed sequence based effects rather than target-directed siRNA total complementarity silencing. Following this discovery, efforts were undertaken and subsequently completed to identify the "true" targets of these clade-specific siRNAs.Item The Function and Mechanism of RNA Interference in Neurospora(2009-01-14) Lee, Heng-Chi; Liu, YiRNA interference (RNAi) is a conserved gene silencing mechanism important for various biological processes, including developmental timing, genome defense, and heterochromatin formation. RNAi is triggered by double stranded RNA (dsRNA), which is processed by Dicer to siRNA. siRNA is loaded onto RNA-induced silencing complex (RISC), in which an Argonaute family protein, guided by a siRNA, mediates the cleavage of homologous RNAs. In the filamentous fungus Neurospora, we show that dsRNA not only trigger RNAi, it also transcriptionally activates several key components of RNAi pathway, including qde-2 (an Argonaute) and dcl-2 (a Dicer). A genome wide identification of dsRNA activated genes suggests that RNAi is part of a broad ancient host-defense response against viral and transposon infections. Our research on qde-2 regulation also suggests a role of RNAi during DNA damage. We show that DNA damage induces qde-2 expression, and the purification of QDE-2 bound RNAs identifies a novel class of small RNAs named qiRNAs. qiRNAs are averaged 21 nt in length and are mostly derived from ribosomal DNA (rDNA) locus. Importantly, qiRNA biogenesis requires RNAi components and RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs during DNA repair. Further analysis suggests that the qiRNA contributes to the DNA damage checkpoints by inhibiting protein translation after DNA damage. To trigger RNAi against transgenes, it has been proposed that transgene- specific aberrant RNA (aRNA) is made and converted into dsRNA by RNA dependent RNA polymerase (RdRP). How aRNA is produced and specifically recognized by RdRP is not known. We show that QDE-1, a RdRP is also the DNA-dependent RNA polymerase (DdRP) that produces aRNA from ssDNA. QDE-1 is recruited to ssDNA by Replication Protein A (RPA) and QDE-3 (an RecQ helicase), both of them are also essential for aRNA production. Moreover, QDE-1 can produce dsRNA from ssDNA, a process facilitated by RPA. Our results provide a molecular mechanism of aRNA production in RNAi pathway.Item Human shRNA Library Screening to Dissect Pathways Involved in Telomerase Actions(2011-12-12) Hoshiyama, Hirotoshi; Shay, Jerry W.The minimal components of human telomerase are the human telomerase reverse transcriptase (hTERT) and the human telomerase template RNA (hTR). Although it is known that both components are minimally sufficient to reconstitute telomerase activity, the factors involved in any of the multiple steps of telomerase action such as telomerase assembly, telomerase recruitment to telomeres, and telomere extension/regulation are not well understood. There are a large numbers of proteins that have associations with telomerase, yet the functional roles of those in telomere maintenance and telomerase regulation are not well understood. Identifying novel proteins and pathways involved in any of these important telomerase-associated functions will be useful for identifying new targets for the development of novel inhibitors that block telomerase function in cancer cells. Therefore, my goal has been to develop methods to dissect these molecular pathways and identify functional factors involved in any step of telomerase actions. To accomplish this, I designed a selective screening system by exploiting lentiviral shRNA libraries and tetracycline inducible-hTERT cell lines that is hTR deficient but expressing mutant hTR. Thus, the overall strategy of the screening system may be considered a “synthetic rescue screen”. In brief this screen was set up to rescue cells from apoptotic death due to mutant sequence incorporation at telomeres by reducing the gene expressions with lentiviral shRNA libraries. This allows us to look a set of genes involved in pathways involved in functional aspects of telomerase actions, not based on structural association with telomerase. During the work, I have established multiple lines of inducible-hTERT cells to use in selective screening systems. I have also developed a method for rapid construction of high-complexity custom shRNA libraries for targeted screening and re-evaluate hundreds of primary candidate genes to identify smaller numbers of secondary candidate genes by removing false positives. In order to analyze the pooled shRNA screening result, I have developed a method for quantitative identification of half-hairpins from a pooled shRNA library based on the pGIPZ vector. Introducing multiplexing codes and refining sample preparation schemes resulted in the predicted ability to detect two-fold enrichments followed by massive parallel sequencing. Development of those methods allowed me to identify several candidate proteins, which may be involved in telomerase actions.Item The Impact of Lipid Nanoparticle Chemistry on RNA Delivery and Therapeutic Outcomes(2022-12-01T06:00:00.000Z) Johnson, Lindsay Taylor; de Gracia Lux, Caroline; Siegwart, Daniel J.; Zhu, Hao; Hoshida, Yujin; Singal, Amit G.This dissertation aims to understand how two individual components of the traditional four-component lipid nanoparticle system, the PEG lipid component and the ionizable cationic lipid component, impact RNA delivery. To systematically investigate how PEG lipid chemistry impacted LNP formulation and RNA delivery, a series of linear-dendritic poly(ethylene glycol) (PEG) lipids were synthesized with modulated hydrophobic domains. The chemical structure of the hydrophobic domain did not impact the formulation of 5A2-SC8 LNPs, including nanoparticle size, RNA encapsulation, and stability. However, the chemical structure did affect RNA delivery efficacy both in vitro and in vivo. The chemical structure of the hydrophobic domain of the PEG lipids impacted the escape of 5A2-SC8 LNPs from endosomes at early cell incubation time points. Overall, the results indicated that PEG lipid anchoring and chemical structure modulated RNA delivery. Although most LNPs accumulate in the liver after intravenous administration (suggesting that liver delivery is straightforward), it was observed that two similar LNP formulations (5A2-SC8 and 3A5-SC14 LNPs) resulted in distinct RNA delivery within the liver organ. Despite both LNPs possessing similar physical properties, the ability to silence RNA in vitro, strong accumulation within the liver, and sharing a pKa of 6.5, only 5A2-SC8 LNPs were able to functionally deliver RNA to hepatocytes. Protein corona analysis indicated that 5A2-SC8 LNPs bind Apolipoprotein E (ApoE), which can drive LDL-R receptor mediated endocytosis in hepatocytes. In contrast, the surface of 3A5-SC14 LNPs was enriched in Albumin but depleted in ApoE, which likely led to Kupffer cell delivery and detargeting of hepatocytes. In an aggressive MYC-driven liver cancer model, 5A2-SC8 LNPs carrying let-7g miRNA were able to significantly extend survival compared the non-treatment group. Since disease targets exist in an organ- and cell-type specific manner, the clinical development of RNA LNP therapeutics will require an improved understanding of LNP cellular tropism within organs. Overall, the results from this work illustrates the importance of understanding the cellular localization of RNA delivery and incorporating further checkpoints when choosing nanoparticles beyond biochemical and physical characterization, as small changes in the chemical composition of LNPs can have an impact on both the biofate of LNPs and therapeutic outcomes.Item The Mechanism of Small Interfering RNA Biogenesis in Neurospora Crassa(2014-01-21) Zhang, Zhenyu; Yu, Hongtao; Liu, Yi; Cobb, Melanie H.; Liu, QinghuaRNA interference is a well-conserved post-transcriptional gene silencing mechanism that regulates various biological processes including development, genome defense, and heterochromatin formation. In filamentous fungus Neurospora crassa, quelling is an RNAi-related phenomenon that post-transcriptionally silences repetitive DNA and transposon. We previously identified a type of DNA damage-induced small RNA called qiRNAs that originate from ribosomal DNA. Ribosomal DNA cluster remains the only highly repetitive sequences in Neurospora genome. To understand how small RNAs are generated from repetitive DNA, we carried out a genetic screen to identify genes required for qiRNA biogenesis. Factors directly involved in homologous recombination (HR) and chromatin remodeling factors required for HR are essential for qiRNA production. HR is also required for quelling, and quelling is also the result of DNA damage, indicating that quelling and qiRNA production share a common mechanism. These results suggest that DNA damage triggered HR-based recombination allows the RNAi pathway to recognize repetitive DNA to produce small RNA. The involvement of chromatin remodeling factors indicates that siRNA biogenesis is regulated in the chromatin level. From our systematic knock out library screen, we identified a novel component, RTT109, which is required for both qiRNA and quelling-induced small RNA production. RTT109 is a fungal-specific histone acetyltransferase (HAT) for histone H3 on lysine 56 and its catalytic activity is required for its function in small RNA production. Furthermore, we show that RTT109 is required for homologous recombination and H3K56Ac is enriched around double strand break, which overlaps with RAD51 binding. Taken together, our results suggest that H3K56 acetylation is required for small RNA production through its role in homologous recombination.Item To Develop a Small Interfering RNA (siRNA) Design and Information Resource to Facilitate Genetic Manipulaton of Human Cells(2004-05-25) Shah, Jyoti Khetsi; Minna, John D.Part I: Small interfering RNAs (siRNAs) have revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In the past, there were a set of rules designed to develop siRNA which worked efficiently in most cases. There was further refinement performed in these rules in some modern research analyses which attempted to address the question of what most closely determines siRNA functionality. I have designed and implemented a new software tool siRNA Information Resource ('sIR') that incorporates the most recent refinements in the design algorithm in order to provide fast and efficient siRNA design. sIR is a web-based computational tool which takes these existing rules for designing synthetic siRNAs and puts them in a software architecture that allows the researcher to design siRNAs for every gene. It also provides a database containing information about already developed siRNA and thus allows the researcher to access the siRNA information database consisting of siRNA information from literature and various other sources. This will ultimately help in future siRNA related discoveries. It also includes a scoring system which helps in rational selection of efficient siRNA. sIR was successfully validated using already designed and developed target siRNA sequences. Part II: One of the major problems in using chemotherapy to treat cancer is whether patients, whose tumors do not respond to one drug, would respond to another. Thus, it would be very useful if one could rationally select the appropriate chemotherapy for each patient's tumor. We are asking is whether tumor gene "expression signatures" detected by microarray analysis could identify a set of genes correlating with sensitivity or resistance to a particular drug. A large panel of breast cancer cell lines was tested with cisplatin, paclitaxel, vinorelbine, doxorubicin and gemcitabine, in vitro using a colorimetric assay to determine the concentration of drug that gives 50% growth inhibition (IC50). Gene expression profiles were also performed using Affymetrix chips and the two data sets were merged. It was found that a panel of ~100 genes were significantly up regulated (4 fold or more) for each drug in resistant cells. As an alternative approach, Pearson correlations between each gene expression data and each drug IC50 across all cell lines analyzed were determined. A positive correlation for a pair of gene and drug indicates the gene may be associated with resistance to the drug whereas a negative correlation would associate that gene with sensitivity to the drug. Some of these genes might be associated with the drug mechanism of action. We conclude that gene expression signatures do exist for individual breast tumor cell chemosensitivity and these could be of clinical significance.Item Using Chemically Modified Oligonucleotides to Modulate Gene Expression, Treat Genetic Diseases, and Probe Novel Mechanisms of RNA Interference(2013-02-20) Yu, Dongbo 1984-; Yu, Hongtao; Liu, Qinghua; Conrad, Nicholas; Corey, David R.A number of inherited neurological disorders remain incurable despite having well-defined monogenic etiologies. One example is Huntington's disease (HD), which is caused by CAG trinucleotide expansion in the gene HUNTINGTIN (HTT) and production of toxic glutamine-expanded protein. Targeting HTT with siRNAs could be a powerful approach, but allele-selectivity is a major challenge: nearly all HD patients are heterozygous at the HTT locus, and expression of wild-type HTT may need to be preserved. One way to achieve allele-selectivity is by exploiting the fact that the mutant HTT allele contains more CAG repeats. Previous work with double-stranded siRNAs (dsRNA) and chemically modified antisense oligonucleotides (ASO) that target the poly-CAG sequence both showed promise but each had significant limitations. To combine the simplicity of ASO and high selectivity of dsRNA, we tested chemically modified, single-stranded small-interfering RNA (ss-siRNA) of sequences targeting CAG repeats in collaboration with ISIS Pharmaceutical, and showed them to have high potency (IC50 ~2 nM) and allele-selectivity (>30-fold) against mutant HTT in HD-patient-derived cell-lines. Mechanistically, CAG-targeting ss-siRNA functions through endogenous RNAi by recruiting Ago2 and GW182 to HTT mRNA in the absence of a passenger strand and reducing mutant HTT protein level without affecting its mRNA level. Selectivity is achieved through preferential cooperative binding of multiple RISC units to the longer poly-CAG tract on the mutant HTT mRNA versus that of the wild-type. Structural-activity relationship (SAR) studies showed that several ss-siRNAs tolerated significant structural modifications and still retained high potency and selectivity. Furthermore, intraventricular infusion of a candidate ss-siRNA in a HD knock-in mouse model yielded selective inhibition of mutant HTT in a wide range of brain regions. Finally, we showed that a subset of ss-siRNAs were also potent, allele-selective inhibitors of ATAXIN-3, the mutated gene in spinocerebellar ataxia type 3 (SCA3). Taken together, we have identified and characterized a novel class of mechanistically interesting and therapeutically promising nucleic-acid-based compounds that could open new doors to finding a cure for genetic diseases such as HD.Item [UT Southwestern Medical Center News](2007-04-11) McKenzie, AlineItem [UT Southwestern Medical Center News](2008-08-11) McKenzie, Aline