Duquesne University Annual Progress Report: 2008 Formula Grant

Size: px

Start display at page:

Download "Duquesne University Annual Progress Report: 2008 Formula Grant"

Annabel Warren
5 years ago
Views:

1 Duquesne University Annual Progress Report: 2008 Formula Grant Reporting Period July 1, 2011 June 30, 2012 Formula Grant Overview The Duquesne University received $94,131 in formula funds for the grant award period January 1, 2009 through June 30, Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose Investigation of the Hepatitis C Virus 3 -Untranslated Region, as a Potential Target for New Antiviral Nucleic-acid Based Strategies - The purpose of this project is to develop novel nucleicacid based therapeutic agents against the Hepatitis C virus (HCV). In the first phase of the project, we will employ fluorescence and NMR spectroscopy to obtain detailed information about the HCV RNA 3 -untranslated (UTR) region, a highly conserved region which has been proposed to be involved in specific interactions with a fragment of the HCV coding region. Genetic data showed that these interactions are absolutely required for the viral replication, making the HCV 3 -UTR region an attractive target for antiviral therapy. In the second phase of the project, we will screen for peptide-nucleic acid aptamers that have the ability to inhibit these essential interactions, and thus, to inhibit the HCV RNA replication. Duration of Project 1/1/2009-6/30/2012 Project Overview Hepatitis C virus (HCV) infection poses a major health problem worldwide, with more than 170 million people being chronically infected. The HCV infection is the most common reason for liver transplantation in the United States; and HCV, together with the hepatitis B virus, is currently responsible for 75% of all cases of liver disease worldwide. No HCV protective vaccine is available to date, making it imperative for new therapeutic strategies to be developed. HCV is a small enveloped virus, whose genome is encoded by a positive strand RNA molecule, which is involved in three essential processes: replication, translation into the viral proteins and packaging into new virions. The RNA genome has an open reading frame and highly conserved 5 - and 3 -untranslated regions (UTRs), which are essential for the virus replication and/or translation. Within the 3 -UTR there is a 98 nucleotide (nt) fragment named X RNA, which is absolutely required for HCV replication. The first 55 nucleotides of X RNA, which are 100% conserved among all HCV strains, contain a 7 nucleotide sequence that has been proposed to be involved in long-range kissing interactions with a novel cis-acting RNA element located within Duquesne University 2008 Formula Grant Page 1

2 the viral RNA polymerase (NS5B) coding sequence. The biological significance of these interactions has been established by showing that mutations that prevent their formation lead to a complete block of the viral replication. This project has the following goals: Specific aim 1. Molecular characterization of the proposed long-range kissing interactions between the HCV 3 -UTR highly conserved X RNA region and the NS5B coding region. Specifically, we will obtain quantitative information about the interactions of the first 55 nt of X RNA with the cis-acting RNA element located in the NS5B coding region by fluorescence spectroscopy. We will also employ NMR spectroscopy to obtain higher resolution structural information about these interactions. Specific aim 2. Screening for nucleic acid based aptamers that block the formation of the long range kissing complex between the HCV 3 -UTR highly conserved X RNA region and the NS5B coding region, preventing the HCV replication. We will design peptide-nucleic acid (PNA) aptamers that will be tested using the fluorescence spectroscopy assays developed under specific aim 1 for the potential to inhibit HCV RNA replication, by inhibiting the interactions between X RNA and the NS5B coding region. Principal Investigator Mihaela Rita Mihailescu, PhD Assistant Professor Duquesne University Department of Chemistry & Biochemistry 600 Forbes Ave. 308 Mellon Hall Pittsburgh, PA, Other Participating Researchers Bruce Armitage, PhD, Danith Ly, PhD - employed by Carnegie Mellon University Sumangala Shetty, BS - employed by Duquesne University Expected Research Outcomes and Benefits While advancing knowledge in the protein-nucleic acids interactions field, this research is specifically important for its potential to lead to the development of new antiviral therapies against the hepatitis C virus. The HCV infection poses a problem globally, as the current treatment, a combination of recombinant alpha interferon and ribavirin, is efficient in less than 50% of the people infected. No HCV protective vaccine is available to date, making it imperative for new therapeutic strategies to be developed. However, the success of these drug discovery efforts depends on the detailed understanding of the molecular processes underlying the HCV life cycle. The results of the current project will provide detailed information about the molecular mechanism of interaction between a highly conserved region of the HCV genomic RNA (3 - UTR) and a cis-acting RNA element located in the HCV coding region, interactions that are essential for the viral replication, and thus, for the HCV life cycle. This knowledge will Duquesne University 2008 Formula Grant Page 2

3 contribute to our ability to develop novel nucleic-acid based antiviral strategies against HCV, by designing peptide nucleic acid aptamers that have the potential to block HCV RNA replication. Summary of Research Completed Specific aim 1. Molecular characterization of the proposed long-range kissing interactions between the HCV 3 -UTR highly conserved X RNA region and the NS5B coding region. The goals of this specific aim have been completed during the previous reporting period. No new experiments were performed during the current reporting period. Specific aim 2. Screening for nucleic acid based aptamers that block the formation of the long range kissing complex between the HCV 3 -UTR highly conserved X RNA region and the NS5B coding region, preventing the HCV replication. In this study we investigated a PNA that was designed against the HCV X RNA sequence , that has been shown to be involved in in vitro RNA homodimerization and also in interactions with the 5BSL3.2 apical loop in the coding region. Given this dual interaction of X RNA, a 10 base PNA was designed against X RNA, such that both the homo- and heterointeractions of X55 RNA with another X55 or with 5BSL3.2 would be disrupted. This PNA was synthesized in the laboratory of Dr. Danith Ly of Carnegie Mellon University. Since this was a first study to investigate the interactions of the PNA with X55 RNA, several experimental conditions such as PNA specificity, temperature, PNA concentration and incubation time were analyzed. i. PNA Specificity: Initially we analyzed the specificity of the PNA developed against X55 RNA. All RNA samples were pretreated with 5 mm MgCl 2 and their interactions with the PNA were analyzed by TBM gel electrophoresis (figure 1). Lanes 1, 2 and 3 in figure 1 contain free 5BSL3.2 RNA, free X55 RNA, and their 1:1 mixture, whereas lanes 4-6 contain identical samples which were incubated for 2 hours at 37 C with 2-fold PNA. When 5BSL3.2 was incubated with PNA (lane 5) a single band corresponding to the monomeric 5BSL3.2 (compare with lane 1) was observed in the gel, indicating the absence of any reactivity of 5BSL3.2 RNA with PNA. However, when the PNA was added to its target X55 RNA (lane 6), the monomer and dimer bands of X55 RNA disappeared, with the concomitant appearance of three new bands (compare with lane 2), which were labeled as Band A (lowermost), Band B (middle) and Band C (topmost). Our results that the PNA binds to X55, but not to 5BSL3.2 indicate that the PNA is specific for X55 RNA. The origin of the bands A, B, and C is not clear, initially we assumed that bands A and B correspond to the PNA bound to the two monomeric conformations of X55 RNA, whereas band C corresponds to a dimer of X55 bound by PNA. However, this hypothesis was not confirmed experimentally, as these bands are also present in the absence of MgCl 2, when X55 exists in a single monomeric conformation (data not shown). Further studies as needed to elucidate the origin of the A, B and C bands. ii. Concentration Dependence: To narrow down the effective required PNA concentration, we treated the target X55 RNA with increasing concentrations of PNA (0.25-fold, 0.5-fold and 1- fold PNA). As seen in figure 2, a single band is present in lane 1 which contains free 5BSL3.2, Duquesne University 2008 Formula Grant Page 3

4 two monomeric and one dimer band are present in lane 2 which contains free X55 RNA. As expected, both the X55-X55 homodimer and the X55-5BSL3.2 heterodimer bands are present in lane 3, as well as some unreacted free 5BSL3.2. As a 0.25-fold PNA is added (figure 2, lane 4), both the monomeric X55 bands and the X55 homodimer band disappear with the concomitant apparition of bands A, B and a faint band C due to the interactions of X55 with PNA. The X55-5BSL3.2 heterodimer band though weaker than in lane 3, is still present in lane 4. In the presence of 0.5-fold PNA (lane 5), the X55-5BSL3.2 heterodimer band gets weaker while the bands A and B become more intense. Finally, upon the addition of 1-fold PNA (lane 6), both the X55 homodimer and the heterodimer bands disappear, while the monomeric 5BSL3.2 band and the bands A, B and C caused due to X55-PNA interactions are more intense. These results clearly indicate that at 37 C, the treatment of X55 RNA with 1-fold PNA for 2 hours is effective in disrupting both, the X55-X55 homodimer, as well as the X55-5BSL3.2 heterodimer interaction. Similar experiments were performed to determine the optimum temperature and time of incubation, as well as PNA concentration (data not shown). Based on these results we can definitely confirm that the PNA we designed is highly specific to its target X55 RNA at concentrations up to 8 fold to target RNA. The efficiency of PNA appears to be similar at both 21 C and 37 C, with as little as 1-fold PNA being efficient in completely disrupting both the homo- and hetero-interactions of X55 in a time period of an hour. In addition, even at lower than 1 fold concentrations, the PNA affected the homo and hetero interactions of X55. iii. Time, Concentration and Temperature: To determine the optimum conditions (concentration, temperature and incubation period) required for PNA to disrupt the X55 interactions, we incubated a preformed kissing complex mixture of X55 and 5BSL3.2 with varying concentrations of PNA (1-6 fold) for 2 hours at two different temperatures: 21 C and 37 C. Figure 3A lane 3 shows the mixture of 5BSL3.2-X55 prior to PNA treatment at 21 C, and as expected, both the homo- and hetero-interaction bands of X55 RNA are clearly visible. Lanes 4-7 contain a preformed kissing complex mixture of 5BSL3.2-X55 which was reacted with increasing concentration of PNA (1-6 fold) at 21 C for 2 hours. It is clear from the lanes 4-7 of figure 3A that even at lower temperature of 21 C, the PNA is efficiently able to disrupt both homo and hetero dimer interaction of X55 RNA. Also, note PNA concentration as little as of 1 fold was proficient at 21 C (Figure 3A lane 4) to disrupt the essential homo and hetero kissing complexes formed by X55 RNA with another X55 or with 5BSL3.2 RNA. Figure 3B shows results for the preformed kissing complex samples of X55 and 5BSL3.2 that were incubated at 37 C with varying concentrations of PNA (1-6 fold) for 2 hrs. We observed very similar results as seen for samples incubated at 21 C. Duquesne University 2008 Formula Grant Page 4

Figure 1: 12% TBM gel in the presence of 5 mm MgCl 2. Lane 1-5BSL3.

2 (15µM) +2- fold PNA (30µM) for 2 hours at 37 C), Lane 5-5BSL3.

at 37 C. Figure 2:12% TBM gel in the presence of 5mM magnesium.

5 Figure 1: 12% TBM gel in the presence of 5 mm MgCl 2. Lane 1-5BSL3.2 (15µM) Lane 2-X55 (15µM), Lane 3-5BSL3.2 (15µM) +X55 (15µM), Lane 4-X55 (15µM) +5BSL3.2 (15µM) +2- fold PNA (30µM) for 2 hours at 37 C), Lane 5-5BSL3.2 (15µM) +2-fold PNA (30µM) for 2 hours at 37 C, Lane 6-X55 (15µM) +2-fold PNA (30µM) for 2 hours at 37 C. Figure 2:12% TBM gel in the presence of 5mM magnesium. All samples were pretreated with magnesium. Lane 1: 5BSL3.2, Lane 2: X55, Lane 3:5BSL3.2+X55, Lanes 4 to 6- X55+5BSL3.2+PNA (0.25 to 1 fold for 2 hours at 37 C). Duquesne University 2008 Formula Grant Page 5

A. B. Figure 3:12% TBM gel in the presence of 5 mm magnesium. All samples were pretreated with magnesium and incubated with the PNA for 2 hours at 21 C (A.) and 37 C (B.), respectively. Lane 1: 5BSL3.

Research Project 2: Project Title and Purpose Impact of Parental Smoking Cessation and Residential Hazard Reduction on Pediatric Respiratory Health: A Pilot Investigation - This two-year pilot

6 A. B. Figure 3:12% TBM gel in the presence of 5 mm magnesium. All samples were pretreated with magnesium and incubated with the PNA for 2 hours at 21 C (A.) and 37 C (B.), respectively. Lane 1: 5BSL3.2, Lane 2: X55, Lane 3:5BSL3.2+X55 in a 1:1 ratio, Lanes 4 to 7 X55+5BSL3.2+PNA (1 fold to 6 fold, as indicated in the figures). Research Project 2: Project Title and Purpose Impact of Parental Smoking Cessation and Residential Hazard Reduction on Pediatric Respiratory Health: A Pilot Investigation - This two-year pilot project involved research-based residential assessments and interventions designed to directly and positively influence the respiratory health of children of smokers by reducing the ambient levels of tobacco smoke and other environmental triggers. A total of 50 families were to have participated over the course of the project. Triggers of primary concern were environmental tobacco smoke and combustion fumes, insect, rodent, pet, and dust mite allergens. Secondary triggers such as mold, dust, and household chemicals were also monitored. Based on the triggers identified in the initial assessment, a plan for parental education intervention with follow-up monitoring of participant respiratory health and ambient hazard levels was created for each family. Duration of Project 1/1/2009-6/30/2012 Project Overview The first objective of this pilot program investigation was to develop local, community-based partnerships among Duquesne University, Healthy Home Resources (a Pittsburgh-based nonprofit), and the community. This program was intended to create the capacity to conduct Duquesne University 2008 Formula Grant Page 6

7 housing and health assessments of the program participants and deliver in-home, communitybased education on asthma and respiratory illnesses and allergen trigger control through a community services worker model. Also included were provisions of standards-based remediation/trigger control protocols and follow-up housing and health assessments that were supported by an exemplary evaluation design. Participants were be recruited from either the Healthy Home Resources AT HOMe (Asthma Trigger HOMe evaluation) program, or by referral from Tobacco Free Allegheny, a local partner in providing education and outreach for smoking cessation. The principal eligibility requirements were that one or both parents or caregivers smoke, and that there were children under age 17 in residence. Various types of assessments were used to conduct the research for the program. The proposed Environmental Assessment was based on American Industrial Hygiene Association protocols. Residential sampling was to have taken place twice, consisting of an initial baseline and postintervention measurements. The proposed Respiratory Health Assessment consisted of a carbon monoxide (CO) breath test for smoking parents and their children. In addition, a self-reporting respiratory illness survey was used that monitored days absent from school, emergency room visits, days with respiratory symptoms, and (for asthmatic children) frequency of rescue medication use. During the initial visit, staff provided the parents or care-givers a Knowledge, Attitudes, and Beliefs Survey (KAB). This tool, designed by colleagues at the University of Pittsburgh Graduate School of Public Health and modeled on the learning objectives in the American Respiratory Alliance curriculum, was used to determine the effectiveness of the educational intervention by comparing the initial results to the follow-up KAB survey. Following the initial KAB survey, general educational materials developed by Healthy Home Resources and Tobacco Free Allegheny were given to the parent(s) or care-givers. These educational resources provided the parents with a general understanding of the risks of second-hand smoke to their children, as well as awareness of other residential triggers of respiratory illness or distress. Based on the initial Environmental and Respiratory Health Assessments, in conjunction with parent/caregiver KAB, an intervention plan was determined for each participating family. Principal Investigator Stanley J. Kabala, PhD Research Professor Duquesne University 600 Forbes Avenue Pittsburgh, PA Other Participating Researchers Michael J. Tobin, PhD, originally Executive Director of Health Home Resources, subsequently employed by Duquesne University on the project Expected Research Outcomes and Benefits A goal of this pilot project was to improve child respiratory health in terms of morbidity and severity. Specifically, it was anticipated that educational outreach to smoking parent(s) and caregiver(s) would improve living conditions in the homes of 50 families in Allegheny County over a Duquesne University 2008 Formula Grant Page 7

8 two-year period. Also, it was expected that an increase in asthma and respiratory illness prevention knowledge by 30% or more (as evidenced by the initial and follow-up KAB surveys) would occur. Additionally, it was anticipated that the program would improve school attendance of participating children by 20% or more and decrease respiratory distress symptom days by an average of 6 days or more. The outcomes of the research component of this project lead to a two-pronged course of action on the efficacy of combining smoking cessation with residential hazard awareness to improve pediatric respiratory health. These outcomes include recommendations for policy change at the local, state, and national levels and improved design and implementation of residential and community intervention and education projects. Summary of Research Completed In June 2011, the Principal and Co- Principal Investigators initiated a no-cost extension request to the Pennsylvania Department of Health that entailed how services were to be provided This request was granted, extending the project term to June 30, The principal programmatic change under the revision was to make Co-Principal Investigator Dr. Michael J. Tobin the main project researcher, based on his specific expertise and his experience as Executive Director of HHR. This was done by placing him under contract with Duquesne University. During this time period we compared the heretofore unpublished AT HOMe intervention outcomes, for which there are over 300 participant families, to published parental smoking cessation/pediatric asthma mitigation studies. Smoking cessation interventions and outcomes for this pilot investigation are identical to the larger AT HOMe cohort, warranting confidence that the pooled AT HOMe outcomes are representative of the pilot results but with a higher degree of statistical power. The service delivery model that we examined has three primary or structural and three secondary or procedural characteristics. The most important primary attribute is that of using a non-profit, community-based organization to deliver health service. That is joined by the use of a research organization to conduct research in the context of that service delivery with the goal of refining and improving the delivery system, and a thorough-going reliance on partnerships with other institutions to ensure the best expertise achieved cost-effectively. Secondary characteristics include appropriate personality and skill of the service delivery personnel, a carefully crafted protocol for both individual home visits and the overall process of visits, and careful attention to cultural sensitivity both in the design of program materials and on the part of personnel. Duquesne University 2008 Formula Grant Page 8

Hepatitis C virus RNA: molecular switches mediated by long-range RNA RNA interactions?

2526 2540 Nucleic Acids Research, 2013, Vol. 41, No. 4 Published online 28 December 2012 doi:10.1093/nar/gks1318 Hepatitis C virus RNA: molecular switches mediated by long-range RNA RNA interactions? Sumangala