Gene therapy has the potential to treat a wide variety of diseases. Delivering nucleic acids, such as DNA and mRNA, allows for the production of an aberrant or absent protein that is causing the disease. Delivery of genes via viruses is very efficient but falls short because of other issues. Nonviral delivery, on the other hand, struggles with efficiency but has advantages in terms of lack of immunogenicity, ease in production, and carrying capacity. DNA is much more stable than mRNA, and the protein production from DNA persists for a longer time. However, DNA delivered to cells must pass through the nuclear envelope to produce protein. Nuclear penetration with nonviral DNA delivery in vivo has not yet been accomplished. mRNA only needs to be delivered to the cytoplasm. Recent interest in nonviral delivery of mRNA has surged upward because delivery of mRNA to various cells in vivo has proven successful. Yet mRNA still struggles with nuclease stability, which is a major impediment toward efficient expression. A polyacridine PEG-peptide (PEG-peptide) has been previously used to stabilize DNA against nuclease hydrolysis by binding through ionic and intercalative interactions. Binding of PEG-peptide to DNA results in a PEGylated nanoparticle, or polyplex, and which protects the DNA. The same PEG-peptide was applied to mRNA. To increase the ability of PEG-peptide to bind through intercalation, a reverse complementary strand was hybridized to the mRNA, forming double stranded mRNA (dsmRNA). In a similar manner to DNA, complexing dsmRNA or single stranded mRNA (ssmRNA) with PEG-peptide resulted in formation of PEG-peptide polyplexes. A dsmRNA polyplex was much more resistant to ribonuclease challenge in vitro than a ssmRNA polyplex. The mRNA constructs were tested in vivo by hydrodynamic dosing. dsmRNA was found to be translationally competent by producing a high level of luciferase reporter enzyme in the liver of mice. When the reverse strand length was modified such that it hybridized with only the coding region, leaving the untranslated regions (UTRs) and poly(A) tail single stranded, the in vivo translatability (level of expression) and persistence (duration of expression) of dsmRNA was equivalent to that of ssmRNA. Full hybridization of the reverse strand with the coding region, the UTRs, and poly(A) tail resulted in a decrease of in vivo translatability. However, the circulatory stability (an in vivo measure of resistance to degradation in blood) was greatly increased when the reverse strand was fully hybridized. The persistence of expression of exogenously delivered mRNA is poor in comparison to DNA. The first step in mRNA decay in the cytoplasm is predominantly poly(A) tail shortening, or deadenylation. To address the persistence issue, mRNA with nonadenosine extensions at the 3’ end of the poly(A) tail was synthesized to inhibit deadenylation-dependent mRNA decay. However, increase of the length of tail extension resulted in a concomitant overall decrease in translatability and no increase in persistence. Hybridization of a DNA oligo to the origin of the tail extension activated endogenous RNase H, cleaving the tail extension, exposing the poly(A) tail, and reactivating the mRNA for high level translation, although no increase in persistence was seen with this strategy. A structured tail extension consisting of two human β-globin 3’UTR sequences increased persistence but also decreased overall translatability. Enzymatic poly(A) tailing of this structured tail extension brought back the translatability but simultaneously lost the persistence gain. While this study on poly(A) tail extension mRNA did not produce a highly active mRNA that had increased persistence, its results may be applicable toward other gene therapy applications. Other efforts to increase the metabolic stability or persistence of mRNA were pursued. Scavenger receptors on resident liver macrophages remove polyplexes from the blood by phagocytosis. Saturation of the scavenger receptors by coadministration of a scavenger receptor inhibitor resulted in increased circulatory stability of dsmRNA. However the scavenger receptor inhibitor was toxic in mice. Another effort to increase the persistence of gene expression in vivo was utilizing an autogene. Autogenes are able to drive the expression of a DNA-based gene outside of the nucleus. In its final form, the autogene did not produce expression. It is an exciting time to be in the field of mRNA gene therapy. Hopefully the research presented in this thesis will factor in to the knowledge base that can treat and cure human diseases.

<p>In Drosophila melanogaster, where males are XY and females are XX, dosage compensation is acheived by approximately two-fold upregulation of transcription of the single male X chromosome. This upregulation is mediated by the dosage compensation complex (DCC), which is assembled in a sequential manner on the male X chromosome and is composed of the two noncoding roX (RNA on the X) RNAs and at least five proteins, including the RNA helicase Maleless (MLE). MLE contains two highly conserved double stranded RNA binding domains (DRBDs) at the N terminus. We investigated the roles of the roX RNAs and MLE helicase through experiments using classical genetic methods to generate and analyze the effects of mutants and mutant transgenes, immunolocalization experiments to study MSL protein and roX RNA to chromosomes. For the first time in vivo, we demonstrate that MLE associates with double stranded RNA in a sequence non-specific manner that is independent of other DCC components. Importantly, we find that the DSRBDs of MLE are essential for dosage compensation but are not required for MLE localization to the male X chromosome. We propose that although the DSRBDs are not essential for ds RNA binding, they may act synergistically with other domains of MLE or other MSLs to associate with RNA in vivo. We propose that a MLE/ roX RNA association involving secondary structure formed by the roX RNAs may be involved in the assembly, stabilization, or function of the DCC.</p> / Dissertation

The study of the DNA has found application beyond our understanding of its cellular function and into a variety of materials assembly and nucleic acid detection systems. The current research investigates double-stranded DNA probes in both a colloidal particle assembly and fluorescent assay format utilizing competitive hybridization events. In both contexts, the affinity of the dsProbes is tuned by the sequence design parameters of duplex length and complementarity. These systems were incubated with nucleic acid targets of interest and, based on the mechanism of competitive hybridization, were responsive to the presence of a high affinity competitive target. In the case of the particle assemblies, incubation with the competitive target resulted in observable disassembly of particle structures. In the case of fluorescently labeled dsProbes, incubation with competitive targets resulted in a quantifiable loss of fluorescence as determined by flow cytometry. Utilizing the fluorescently labeled dsProbe system, the kinetics of competitive hybridization was characterized for nucleic acid targets of varying specificity and strand context. The results indicate promise for the development of the competitive hybridization approach in nucleic acid detection systems providing advantages over current single-stranded probe designs. By utilizing a fluorescently labeled dsProbe approach, it is unnecessary to chemically modify the target of interest to impart a signaling mechanism. Additionally, as the process of competitive hybridization of dsProbes with targets of interest is an affinity driven process, discrimination of targets based on specificity is decoupled from standard measures such as elevated temperature protocols, an important step in translating nucleic acid technologies from the controlled laboratory environment to field applications.

DNA double strand breaks (DSB) are the most toxic of all types of DNA lesions. In Saccharomyces cerevisiae, DNA DSBs are predominantly repaired by the homologous recombination repair (HRR) pathway. The initial step of HRR requires extensive processing of DNA ends from the 5' to 3' direction by specific endo-exonuclease(s) (EE) at the DSB sites, but no endo-exonuclease(s) has yet been conclusively determined for such processing of DSBs. S. cerevisiae TRM2/RNC1 is a candidate endo-exonuclease that was previously implicated for its role in the HRR pathway and was also shown to have methyl transferase activity primarily located at its c-terminus. / In this dissertation, we provided compelling biochemical and genetic evidence that linked TRM2/RNC1 to the DNA end processing role in HRR. Trm2/Rnc1p purified with a small calmodulin binding peptide (CBP) tag displayed single strand (ss) specific endonuclease and double strand (ds) specific 5' to 3' exonuclease activity characteristic of endo-exonucleases involved in HRR. Intriguingly, purified Trm2/Rnc1p deleted for its C-terminal methyl transferase domain retained its nuclease activity but not the methyl transferase activity indicating that the C-terminal part responsible for its methyl transferase function is not required for its nuclease activity. / Our genetic and functional studies with S. cerevisiae trm2/rnc1 single mutants alone or in combination with other DNA DSB repair mutants after treatment with the DNA damaging drug methyl methane sulfonate (MMS) or IR that is believed to produce DSBs, or with specific induction of DNA DSBs at the MAT locus by HO-endonuclease demonstrated an epistatic relationship of TRM2/RNC1 with the major recombination factor RAD52. These studies suggested that TRM2/RNC1 probably acts at an earlier step than RAD52 in the HRR pathway. The genetic evidence also indicated a possible functional redundancy with the bona fide endo-exonuclease EXO1 in the processing of DNA ends at the DSB sites. / In a recent report, the immuno-purified mouse homologue of TRM2/RNC1 exhibited similar enzymatic properties as the endo-exonucleases involved in HRR. A small molecular inhibitor pentamidine specifically inhibited the nuclease activity of the mouse EE and sensitized various cancer cells to DNA damaging agents commonly used in cancer chemotherapy. We specifically suppressed expression of the mouse EE using small interfering RNA (siRNA) that conferred sensitivity of B16F10 melanoma cells to a variety of DNA damaging drugs often used in cancer treatment. This further validated our earlier claim of the endo-exonuclease as a potential therapeutic target in treating cancer.

Viral double-stranded RNA is a key pathogen invasion signal recognized by the human innate immune system. All adenoviruses synthesize at least one highly structured RNA (VAI) to suppress this antiviral response by attenuating the activity of antiviral proteins. Surprisingly, VAI RNA was previously shown to positively regulate the activity of one interferon-inducible antiviral protein, 2’-5’-oligoadenylate synthetases (OAS). The present thesis focuses on investigating the regulation of a human OAS1 isoform by VAI RNA and its derivatives. An Escherichia coli protein expression and purification system has been developed for OAS1 protein production. A combination of biochemical and biophysical approaches was employed to examine VAI RNA binding affinity, activation potential for OAS1 and OAS1:VAI RNA complex formation. Taken together, I have found that while full-length VAI does indeed activate OAS1 in vitro, a truncated version lacking the terminal stem has the opposite effect, and this is the physiologically important response.

Viral double-stranded RNA is a key pathogen invasion signal recognized by the human innate immune system. All adenoviruses synthesize at least one highly structured RNA (VAI) to suppress this antiviral response by attenuating the activity of antiviral proteins. Surprisingly, VAI RNA was previously shown to positively regulate the activity of one interferon-inducible antiviral protein, 2’-5’-oligoadenylate synthetases (OAS). The present thesis focuses on investigating the regulation of a human OAS1 isoform by VAI RNA and its derivatives. An Escherichia coli protein expression and purification system has been developed for OAS1 protein production. A combination of biochemical and biophysical approaches was employed to examine VAI RNA binding affinity, activation potential for OAS1 and OAS1:VAI RNA complex formation. Taken together, I have found that while full-length VAI does indeed activate OAS1 in vitro, a truncated version lacking the terminal stem has the opposite effect, and this is the physiologically important response.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 126-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (M.Sc.). / Written for the Division of Experimental Medicine. Title from title page of PDF (viewed 2009/06/19). Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Molecular Virology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...