Publications by authors named "Bret Heale"

21 Publications

  • Page 1 of 1

vcf2fhir: a utility to convert VCF files into HL7 FHIR format for genomics-EHR integration.

BMC Bioinformatics 2021 Mar 2;22(1):104. Epub 2021 Mar 2.

Brigham and Women's Hospital, Boston, MA, USA.

Background: VCF formatted files are the lingua franca of next-generation sequencing, whereas HL7 FHIR is emerging as a standard language for electronic health record interoperability. A growing number of FHIR-based clinical genomics applications are emerging. Here, we describe an open source utility for converting variants from VCF format into HL7 FHIR format.

Results: vcf2fhir converts VCF variants into a FHIR Genomics Diagnostic Report. Conversion translates each VCF row into a corresponding FHIR-formatted variant in the generated report. In scope are simple variants (SNVs, MNVs, Indels), along with zygosity and phase relationships, for autosomes, sex chromosomes, and mitochondrial DNA. Input parameters include VCF file and genome build ('GRCh37' or 'GRCh38'); and optionally a conversion region that indicates the region(s) to convert, a studied region that lists genomic regions studied by the lab, and a non-callable region that lists studied regions deemed uncallable by the lab. Conversion can be limited to a subset of VCF by supplying genomic coordinates of the conversion region(s). If studied and non-callable regions are also supplied, the output FHIR report will include 'region-studied' observations that detail which portions of the conversion region were studied, and of those studied regions, which portions were deemed uncallable. We illustrate the vcf2fhir utility via two case studies. The first, 'SMART Cancer Navigator', is a web application that offers clinical decision support by linking patient EHR information to cancerous gene variants. The second, 'Precision Genomics Integration Platform', intersects a patient's FHIR-formatted clinical and genomic data with knowledge bases in order to provide on-demand delivery of contextually relevant genomic findings and recommendations to the EHR.

Conclusions: Experience to date shows that the vcf2fhir utility can be effectively woven into clinically useful genomic-EHR integration pipelines. Additional testing will be a critical step towards the clinical validation of this utility, enabling it to be integrated in a variety of real world data flow scenarios. For now, we propose the use of this utility primarily to accelerate FHIR Genomics understanding and to facilitate experimentation with further integration of genomics data into the EHR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12859-021-04039-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923512PMC
March 2021

Functional Transcription Factor Target Networks Illuminate Control of Epithelial Remodelling.

Cancers (Basel) 2020 Sep 30;12(10). Epub 2020 Sep 30.

MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.

Cell identity is governed by gene expression, regulated by transcription factor (TF) binding at cis-regulatory modules. Decoding the relationship between TF binding patterns and gene regulation is nontrivial, remaining a fundamental limitation in understanding cell decision-making. We developed the NetNC software to predict functionally active regulation of TF targets; demonstrated on nine datasets for the TFs Snail, Twist, and modENCODE Highly Occupied Target (HOT) regions. Snail and Twist are canonical drivers of epithelial to mesenchymal transition (EMT), a cell programme important in development, tumour progression and fibrosis. Predicted "neutral" (non-functional) TF binding always accounted for the majority (50% to 95%) of candidate target genes from statistically significant peaks and HOT regions had higher functional binding than most of the Snail and Twist datasets examined. Our results illuminated conserved gene networks that control epithelial plasticity in development and disease. We identified new gene functions and network modules including crosstalk with notch signalling and regulation of chromatin organisation, evidencing networks that reshape Waddington's epigenetic landscape during epithelial remodelling. Expression of orthologous functional TF targets discriminated breast cancer molecular subtypes and predicted novel tumour biology, with implications for precision medicine. Predicted invasion role were validated using a tractable cell model, supporting our approach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12102823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652213PMC
September 2020

FHIR Genomics: enabling standardization for precision medicine use cases.

NPJ Genom Med 2020 18;5:13. Epub 2020 Mar 18.

9Departments of Biomedical Informatics and Medicine, Vanderbilt University, Nashville, TN USA.

The development of Fast Healthcare Interoperability Resources (FHIR) Genomics, a feasible and efficient method for exchanging complex clinical genomic data and interpretations, is described. FHIR Genomics is a subset of the emerging Health Level 7 FHIR standard and targets data from increasingly available technologies such as next-generation sequencing. Much care and integration of feedback have been taken to ease implementation, facilitate wide-scale interoperability, and enable modern app development toward a complete precision medicine standard. A new use case, the integration of the Variant Interpretation for Cancer Consortium (VICC) "meta-knowledgebase" into a third-party application, is described.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41525-020-0115-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080712PMC
March 2020

Lessons Learned in Creating Interoperable Fast Healthcare Interoperability Resources Profiles for Large-Scale Public Health Programs.

Appl Clin Inform 2019 01 6;10(1):87-95. Epub 2019 Feb 6.

Department of Biomedical Informatics, Intermountain Healthcare, Murray, Utah, United States.

Objective: This article describes lessons learned from the collaborative creation of logical models and standard Health Level Seven (HL7) Fast Healthcare Interoperability Resources (FHIR) profiles for family planning and reproductive health. The National Health Service delivery program will use the FHIR profiles to improve federal reporting, program monitoring, and quality improvement efforts.

Materials And Methods: Organizational frameworks, work processes, and artifact testing to create FHIR profiles are described.

Results: Logical models and FHIR profiles for the Family Planning Annual Report 2.0 dataset have been created and validated.

Discussion: Using clinical element models and FHIR to meet the needs of a real-world use case has been accomplished but has also demonstrated the need for additional tooling, terminology services, and application sandbox development.

Conclusion: FHIR profiles may reduce the administrative burden for the reporting of federally mandated program data.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0038-1677527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365290PMC
January 2019

Optimization of infobutton design and Implementation: A systematic review.

J Biomed Inform 2017 10 22;74:10-19. Epub 2017 Aug 22.

Department of Biomedical Informatics, University of Utah, Salt Lake City, UT, United States. Electronic address:

Objective: Infobuttons are clinical decision tools embedded in the electronic health record that attempt to link clinical data with context sensitive knowledge resources. We systematically reviewed technical approaches that contribute to improved infobutton design, implementation and functionality.

Methods: We searched databases including MEDLINE, EMBASE, and the Cochrane Library database from inception to March 1, 2016 for studies describing the use of infobuttons. We selected full review comparative studies, usability studies, and qualitative studies examining infobutton design and implementation. We abstracted usability measures such as user satisfaction, impact, and efficiency, as well as prediction accuracy of infobutton content retrieval algorithms and infobutton adoption/interoperability.

Results: We found 82 original research studies on infobuttons. Twelve studies met criteria for detailed abstraction. These studies investigated infobutton interoperability (1 study); tools to help tailor infobutton functionality (1 study); interventions to improve user experience (7 studies); and interventions to improve content retrieval by improving prediction of relevant knowledge resources and information needs (3 studies). In-depth interviews with implementers showed the Health Level Seven (HL7) Infobutton standard to be simple and easy to implement. A usability study demonstrated the feasibility of a tool to help medical librarians tailor infobutton functionality. User experience studies showed that access to resources with which users are familiar increased user satisfaction ratings; and that links to specific subsections of drug monographs increased information seeking efficiency. However, none of the user experience improvements led to increased usage uptake. Recommender systems based on machine learning algorithms outperformed hand-crafted rules in the prediction of relevant resources and clinicians' information needs in a laboratory setting, but no studies were found using these techniques in clinical settings. Improved content indexing in one study led to improved content retrieval across three health care organizations.

Conclusion: Best practice technical approaches to ensure optimal infobutton functionality, design and implementation remain understudied. The HL7 Infobutton standard has supported wide adoption of infobutton functionality among clinical information systems and knowledge resources. Limited evidence supports infobutton enhancements such as links to specific subtopics, configuration of optimal resources for specific tasks and users, and improved indexing and content coverage. Further research is needed to investigate user experience improvements to increase infobutton use and effectiveness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbi.2017.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641258PMC
October 2017

Physicians' pharmacogenomics information needs and seeking behavior: a study with case vignettes.

BMC Med Inform Decis Mak 2017 Aug 1;17(1):113. Epub 2017 Aug 1.

Department of Biomedical Informatics, University of Utah, 421 Wakara Way, Salt Lake City, UT, 84108, USA.

Background: Genetic testing, especially in pharmacogenomics, can have a major impact on patient care. However, most physicians do not feel that they have sufficient knowledge to apply pharmacogenomics to patient care. Online information resources can help address this gap. We investigated physicians' pharmacogenomics information needs and information-seeking behavior, in order to guide the design of pharmacogenomics information resources that effectively meet clinical information needs.

Methods: We performed a formative, mixed-method assessment of physicians' information-seeking process in three pharmacogenomics case vignettes. Interactions of 6 physicians' with online pharmacogenomics resources were recorded, transcribed, and analyzed for prominent themes. Quantitative data included information-seeking duration, page navigations, and number of searches entered.

Results: We found that participants searched an average of 8 min per case vignette, spent less than 30 s reviewing specific content, and rarely refined search terms. Participants' information needs included a need for clinically meaningful descriptions of test interpretations, a molecular basis for the clinical effect of drug variation, information on the logistics of carrying out a genetic test (including questions related to cost, availability, test turn-around time, insurance coverage, and accessibility of expert support).Also, participants sought alternative therapies that would not require genetic testing.

Conclusion: This study of pharmacogenomics information-seeking behavior indicates that content to support their information needs is dispersed and hard to find. Our results reveal a set of themes that information resources can use to help physicians find and apply pharmacogenomics information to the care of their patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12911-017-0510-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540399PMC
August 2017

Integrating Genomic Resources with Electronic Health Records using the HL7 Infobutton Standard.

Appl Clin Inform 2016 08 31;7(3):817-31. Epub 2016 Aug 31.

Bret S.E. Heale, Ph.D., 421 Wakara Way #140, Salt Lake City, UT 84108, Email:

Background: The Clinical Genome Resource (ClinGen) Electronic Health Record (EHR) Workgroup aims to integrate ClinGen resources with EHRs. A promising option to enable this integration is through the Health Level Seven (HL7) Infobutton Standard. EHR systems that are certified according to the US Meaningful Use program provide HL7-compliant infobutton capabilities, which can be leveraged to support clinical decision-making in genomics.

Objectives: To integrate genomic knowledge resources using the HL7 infobutton standard. Two tactics to achieve this objective were: (1) creating an HL7-compliant search interface for ClinGen, and (2) proposing guidance for genomic resources on achieving HL7 Infobutton standard accessibility and compliance.

Methods: We built a search interface utilizing OpenInfobutton, an open source reference implementation of the HL7 Infobutton standard. ClinGen resources were assessed for readiness towards HL7 compliance. Finally, based upon our experiences we provide recommendations for publishers seeking to achieve HL7 compliance.

Results: Eight genomic resources and two sub-resources were integrated with the ClinGen search engine via OpenInfobutton and the HL7 infobutton standard. Resources we assessed have varying levels of readiness towards HL7-compliance. Furthermore, we found that adoption of standard terminologies used by EHR systems is the main gap to achieve compliance.

Conclusion: Genomic resources can be integrated with EHR systems via the HL7 Infobutton standard using OpenInfobutton. Full compliance of genomic resources with the Infobutton standard would further enhance interoperability with EHR systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4338/ACI-2016-04-RA-0058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052552PMC
August 2016

Context-sensitive decision support (infobuttons) in electronic health records: a systematic review.

J Am Med Inform Assoc 2017 Mar;24(2):460-468

Department of Biomedical Informatics, University of Utah, Salt Lake City, UT, USA.

Objective: Infobuttons appear as small icons adjacent to electronic health record (EHR) data (e.g., medications, diagnoses, or test results) that, when clicked, access online knowledge resources tailored to the patient, care setting, or task. Infobuttons are required for "Meaningful Use" certification of US EHRs. We sought to evaluate infobuttons' impact on clinical practice and identify features associated with improved outcomes.

Methods: We conducted a systematic review, searching MEDLINE, EMBASE, and other databases from inception to July 6, 2015. We included and cataloged all original research in any language describing implementation of infobuttons or other context-sensitive links. Studies evaluating clinical implementations with outcomes of usage or impact were reviewed in greater detail. Reviewers worked in duplicate to select articles, evaluate quality, and abstract information.

Results: Of 599 potential articles, 77 described infobutton implementation. The 17 studies meriting detailed review, including 3 randomized trials, yielded the following findings. Infobutton usage frequency ranged from 0.3 to 7.4 uses per month per potential user. Usage appeared to be influenced by EHR task. Five studies found that infobuttons are used less often than non-context-sensitive links (proportionate usage 0.20-0.34). In 3 studies, users answered their clinical question in > 69% of infobutton sessions. Seven studies evaluated alternative approaches to infobutton design and implementation. No studies isolated the impact of infobuttons on objectively measured patient outcomes.

Conclusions: Weak evidence suggests that infobuttons can help providers answer clinical questions. Research on optimal infobutton design and implementation, and on the impact on patient outcomes and provider behaviors, is needed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jamia/ocw104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080678PMC
March 2017

RNA-sequencing analysis of 5' capped RNAs identifies many new differentially expressed genes in acute hepatitis C virus infection.

Viruses 2012 04 16;4(4):581-612. Epub 2012 Apr 16.

Department of Medicine, University of Utah, 30 N 1900 E #3C310, Salt Lake City, UT 84132, USA.

We describe the first report of RNA sequencing of 5' capped (Pol II) RNAs isolated from acutely hepatitis C virus (HCV) infected Huh 7.5 cells that provides a general approach to identifying differentially expressed annotated and unannotated genes that participate in viral-host interactions. We identified 100, 684, and 1,844 significantly differentially expressed annotated genes in acutely infected proliferative Huh 7.5 cells at 6, 48, and 72 hours, respectively (fold change ≥ 1.5 and Bonferroni adjusted p-values < 0.05). Most of the differentially expressed genes (>80%) and biological pathways (such as adipocytokine, Notch, Hedgehog and NOD-like receptor signaling) were not identified by previous gene array studies. These genes are critical components of host immune, inflammatory and oncogenic pathways and provide new information regarding changes that may benefit the virus or mediate HCV induced pathology. RNAi knockdown studies of newly identified highly upregulated FUT1 and KLHDC7B genes provide evidence that their gene products regulate and facilitate HCV replication in hepatocytes. Our approach also identified novel Pol II unannotated transcripts that were upregulated. Results further identify new pathways that regulate HCV replication in hepatocytes and suggest that our approach will have general applications in studying viral-host interactions in model systems and clinical biospecimens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v4040581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347324PMC
April 2012

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA.

Biochimie 2012 Jul 23;94(7):1499-509. Epub 2011 Dec 23.

Institute of Molecular Biology and Biophysics, ETH Zurich, Schafmattstrasse 20, CH-8093 Zürich, Switzerland.

Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2011.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539133PMC
July 2012

The Effect of RNA Editing and ADARs on miRNA Biogenesis and Function.

Adv Exp Med Biol 2011 ;700:76-84

, .

From analysis of deep-sequencing data it is apparent that sequence differences occur between the genome and miRNAs. Changes from genomic A to an apparent G in miRNA can be accounted for by the editing activity of ADARs. Questions that arise from this observation are: How many miRNAs are edited and to what frequency? Is there a specific step in the biogenesis of miRNAs that is preferentially susceptible to editing by ADARs? However the key question is whether editing affects the downstream activity of miRNAs. Despite much evidence that miRNAs are edited, critical examination of the functional data shows a dearth of examples where editing has been demonstrated to actually affect the downstream miRNA activity in vivo. Even where it is demonstrated that RNA editing can affect biogenesis or targeting of a particular miRNA, effects may be limited by redundancy within the miRNA network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4419-7823-3_8DOI Listing
April 2016

Analysis of A to I editing of miRNA in macrophages exposed to Salmonella.

RNA Biol 2010 Sep-Oct;7(5):621-7. Epub 2010 Sep 1.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, UK.

The main mediator of the lipopolysaccharide (LPS) response in macrophages is activation of Toll-like receptor 4 (TLR4). This generates interferon-beta (INF-beta) production that stimulates increased expression of the RNA editing enzyme ADAR1. To determine if there is an increase in RNA editing in mature miRNA in response to TLR4 activation upon Salmonella infection of macrophages we analyzed small RNA deep sequencing data. Interestingly, we found that direct infection of macrophage cell lines with Salmonella does not result in an increase of edited mature miRNA. Thus, despite elevated levels of ADAR1 during TLR4 activation of macrophages mediated by Salmonella infection, ADAR1 does not result in redirection of miRNA. The most common consequence of ADAR activity on miRNA is a reduction in the mature miRNA level due to interference with miRNA processing of pri-miRNA. However, we found very few miRNAs with reductions in level, and no significant difference between miRNAs previously reported to be edited and those reported to be not edited. In particular, we did not see significant decrease in mir-22 and mir-142, nor editing of pri-mir-22 or pri-mir-142 in infected RAW macrophages. Thus, ADAR1 has very little, if any, effect on the miRNA machinery following TL4 activation by Salmonella infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073258PMC
http://dx.doi.org/10.4161/rna.7.5.13269DOI Listing
June 2011

A role for human Dicer in pre-RISC loading of siRNAs.

Nucleic Acids Res 2011 Mar 23;39(4):1510-25. Epub 2010 Oct 23.

Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010, USA.

RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkq846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045585PMC
March 2011

The effect of RNA editing and ADARs on miRNA biogenesis and function.

Adv Exp Med Biol 2010 ;700:76-84

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

From analysis of deep-sequencing data it is apparent that sequence differences occur between the genome and miRNAs. Changes from genomic A to an apparent G in miRNA can be accounted for by the editing activity of ADARs. Questions that arise from this observation are: How many miRNAs are edited and to what frequency? Is there a specific step inthebiogenesis of miRNAs that is preferentially susceptible to editing by ADARs? However the key question is whether editing affects the downstream activity ofmiRNAs. Despite much evidence that miRNAs are edited, critical examination of the functional data shows a dearth of examples where editing has been demonstrated to actually affect the downstream miRNA activity in vivo. Even where it is demonstratedthat RNA editing can affect biogenesis or targeting of a particular miRNA, effects may be limited by redundancy within the miRNA network.
View Article and Find Full Text PDF

Download full-text PDF

Source
June 2011

ADARs have effects beyond RNA editing.

Cell Cycle 2009 Dec 30;8(24):4011-2. Epub 2009 Dec 30.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.8.24.10214DOI Listing
December 2009

Editing independent effects of ADARs on the miRNA/siRNA pathways.

EMBO J 2009 Oct 27;28(20):3145-56. Epub 2009 Aug 27.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, UK.

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emboj.2009.244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735678PMC
October 2009

Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo.

Clin Cancer Res 2007 Apr;13(7):2207-15

Calando Pharmaceuticals, Inc., California Institute of Technology, Pasadena, California 91107, USA.

Purpose: Ribonucleotide reductase (RR) is a therapeutic target for DNA replication-dependent diseases such as cancer. Here, a potent small interfering RNA (siRNA) duplex against the M2 subunit of RR (RRM2) is developed and shown to reduce the growth potential of cancer cells both in vitro and in vivo.

Experimental Design: Three anti-RRM2 siRNAs were identified via computational methods, and the potency of these and additional "tiling" duplexes was analyzed in cultured cells via cotransfections using a RRM2-luciferase fusion construct. Knockdown of RRM2 by the best duplex candidates was confirmed directly by Western blotting. The effect of potent duplexes on cell growth was investigated by a real-time cell electronic sensing assay. Finally, duplex performance was tested in vivo in luciferase-expressing cells via whole animal bioluminescence imaging.

Results: Moderate anti-RRM2 effects are observed from the three duplexes identified by computational methods. However, the tiling experiments yielded an extremely potent duplex (siR2B+5). This duplex achieves significant knockdown of RRM2 protein in cultured cells and has pronounced antiproliferative activity. S.c. tumors of cells that had been transfected with siR2B+5 preinjection grew slower than those of control cells.

Conclusions: An anti-RRM2 siRNA duplex is identified that exhibits significant antiproliferative activity in cancer cells of varying human type and species (mouse, rat, monkey); these findings suggest that this duplex is a promising candidate for therapeutic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-06-2218DOI Listing
April 2007

Distance constraints between microRNA target sites dictate efficacy and cooperativity.

Nucleic Acids Res 2007 27;35(7):2333-42. Epub 2007 Mar 27.

Division of Molecular Biology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.

MicroRNAs (miRNAs) have the potential to regulate the expression of thousands of genes, but the mechanisms that determine whether a gene is targeted or not are poorly understood. We studied the genomic distribution of distances between pairs of identical miRNA seeds and found a propensity for moderate distances greater than about 13 nt between seed starts. Experimental data show that optimal down-regulation is obtained when two seed sites are separated by between 13 and 35 nt. By analyzing the distance between seed sites of endogenous miRNAs and transfected small interfering RNAs (siRNAs), we also find that cooperative targeting of sites with a separation in the optimal range can explain some of the siRNA off-target effects that have been reported in the literature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkm133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1874663PMC
June 2007

Metalloproteases regulate T-cell proliferation and effector function via LAG-3.

EMBO J 2007 Jan;26(2):494-504

Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases approximately 12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCtheta-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3(-/-) T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.emboj.7601520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783452PMC
January 2007

siRNA target site secondary structure predictions using local stable substructures.

Nucleic Acids Res 2005 Feb 18;33(3):e30. Epub 2005 Feb 18.

Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope Duarte, CA 91010, USA.

The crystal structure based model of the catalytic center of Ago2 revealed that the siRNA and the mRNA must be able to form an A-helix for correct positing of the scissile phosphate bond for cleavage in RNAi. This suggests that base pairing of the target mRNA with itself, i.e. secondary structure, must be removed before cleavage. Early on in the siRNA design, GC-rich target sites were avoided because of their potential to be involved in strong secondary structure. It is still unclear how important a factor mRNA secondary structure is in RNAi. However, it has been established that a difference in the thermostability of the ends of an siRNA duplex dictate which strand is loaded into the RNA-induced silencing complex. Here, we use a novel secondary structure prediction method and duplex-end differential calculations to investigate the importance of a secondary structure in the siRNA design. We found that the differential duplex-end stabilities alone account for functional prediction of 60% of the 80 siRNA sites examined, and that secondary structure predictions improve the prediction of site efficacy. A total of 80% of the non-functional sites can be eliminated using secondary structure predictions and duplex-end differential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gni026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC549425PMC
February 2005

Rapid assessment of anti-HIV siRNA efficacy using PCR-derived Pol III shRNA cassettes.

Mol Ther 2004 Sep;10(3):597-603

Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.

Identification of sequences within a target mRNA that are susceptible to potent siRNA knockdown often requires testing several independent siRNAs or shRNA expression cassettes. Using RNAi against HIV RNAs is further complicated by the length of the viral genome, the complexity of splicing patterns, and the propensity for genetic heterogeneity; consequently, it is most important to identify a number of siRNA targets that potently block viral replication. We previously described a facile PCR-based strategy for rapid synthesis of si/shRNA expression units and their testing in mammalian cells. Using this approach, which is rapid and inexpensive, it is possible to screen a number of potential RNAi targets in HIV to identify those that are most susceptible to RNAi. We report that shRNA expression cassettes constructed by PCR and cotransfected directly into mammalian cells with HIV proviral DNA express shRNAs that are inhibitory to HIV-1 replication. Our results also demonstrate that there is a wide range of efficacies among shRNAs targeting different sites throughout the HIV genome. By screening several different targets we were able to identify a sequence in a common tat/rev exon that is exquisitely sensitive to RNAi. Furthermore we relate the efficacies of our PCR product expressed shRNAs to the relative stabilities of the siRNA duplexes and the accessibilities of the target sites to antisense base pairing in cell extracts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2004.05.003DOI Listing
September 2004
-->