Overcoming Barriers for Sirna Therapeutics: from Bench to Bedside
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Patenting Interfering RNA
Patenting Interfering RNA J. Douglas Schultz SPE Art Unit 1635 (571) 272-0763 [email protected] Oligonucleotide Inhibitors: Mechanisms of Action RNAi - Mechanism of Action • dsRNA induces sequence-specific degradation of homologous gene transcripts • RISC metabolizes dsRNA to small 21-23- nucleotide siRNAs – RISC contains dsRNase (“Dicer”), ssRNase (Argonaut 2 or Ago2) • RISC utilizes antisense strand as “guide” to find cleavable target siRNA Mechanism of Action miRNA Mechanism of Action Interfering RNA Glossary of Terms • RNAi – RNA interference • dsRNA – double stranded RNA • siRNA – small interfering RNA, double stranded, 21-23 nucleotides • shRNA – short hairpin RNA (doubled stranded by virtue of a ssRNA folding back on itself) • miRNA – micro RNA • RISC – RNA-induced silencing complex – Dicer – RNase endonuclease siRNA miRNA • Exogenously delivered • Endogenously produced • 21-23mer dsRNA • 21-23mer dsRNA • Acts through RISC • Acts through RISC • Induces homologous target • Induces homologous target cleavage cleavage • Perfect sequence match • Imperfect sequence match – Results in target degradation – Results in translation arrest RNAi Patentability issues Sample Claims: • A siRNA that inhibits expression of a nucleic acid encoding protein X. OR • A siRNA comprising a 2’-modification, wherein said modification comprises 2’-fluoro, 2’-O-methyl, or 2’- deoxy. (Note: no target recited) OR • A method of reducing tumor cell growth comprising administering siRNA targeting protein X. RNAi Patentability Issues 35 U.S.C. 101 – Utility • Credible/Specific/Substantial/Well Established. • Used to attempt modulation of gene expression in human diseases • Routinely investigate gene function in a high throughput fashion or to (see Rana RT, Nat. Rev. Mol. Cell Biol. 2007, Vol. 8:23-36). -
Depletion of Kcnq1ot1 Non-Coding RNA Does Not Affect Imprinting Maintenance in Stem Cells Michael C
DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 3667 Development 138, 3667-3678 (2011) doi:10.1242/dev.057778 © 2011. Published by The Company of Biologists Ltd Depletion of Kcnq1ot1 non-coding RNA does not affect imprinting maintenance in stem cells Michael C. Golding1,2,3,*,†, Lauren S. Magri1,2,3,†, Liyue Zhang2,3, Sarah A. Lalone1,2,3, Michael J. Higgins4 and Mellissa R. W. Mann1,2,3,‡ SUMMARY To understand the complex regulation of genomic imprinting it is important to determine how early embryos establish imprinted gene expression across large chromosomal domains. Long non-coding RNAs (ncRNAs) have been associated with the regulation of imprinting domains, yet their function remains undefined. Here, we investigated the mouse Kcnq1ot1 ncRNA and its role in imprinted gene regulation during preimplantation development by utilizing mouse embryonic and extra-embryonic stem cell models. Our findings demonstrate that the Kcnq1ot1 ncRNA extends 471 kb from the transcription start site. This is significant as it raises the possibility that transcription through downstream genes might play a role in their silencing, including Th, which we demonstrate possesses maternal-specific expression during early development. To distinguish between a functional role for the transcript and properties inherent to transcription of long ncRNAs, we employed RNA interference-based technology to deplete Kcnq1ot1 transcripts. We hypothesized that post-transcriptional depletion of Kcnq1ot1 ncRNA would lead to activation of normally maternal-specific protein-coding genes on the paternal chromosome. Post-transcriptional short hairpin RNA-mediated depletion in embryonic stem, trophoblast stem and extra-embryonic endoderm stem cells had no observable effect on the imprinted expression of genes within the domain, or on Kcnq1ot1 imprinting center DNA methylation, although a significant decrease in Kcnq1ot1 RNA signal volume in the nucleus was observed. -
(12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub
US 20040O86860A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub. Date: May 6, 2004 (54) METHODS OF PRODUCING RNAS OF Publication Classification DEFINED LENGTH AND SEQUENCE (51) Int. Cl." .............................. C12Q 1/68; C12P 19/34 (76) Inventor: Muhammad Sohail, Marston (GB) (52) U.S. Cl. ............................................... 435/6; 435/91.2 Correspondence Address: MINTZ, LEVIN, COHN, FERRIS, GLOWSKY (57) ABSTRACT AND POPEO, PC. ONE FINANCIAL CENTER Methods of making RNA duplexes and single-stranded BOSTON, MA 02111 (US) RNAS of a desired length and Sequence based on cleavage of RNA molecules at a defined position, most preferably (21) Appl. No.: 10/264,748 with the use of deoxyribozymes. Novel deoxyribozymes capable of cleaving RNAS including a leader Sequence at a (22) Filed: Oct. 4, 2002 Site 3' to the leader Sequence are also described. Patent Application Publication May 6, 2004 Sheet 1 of 2 US 2004/0086860 A1 DNA Oligonucleotides T7 Promoter -TN-- OR 2N-2-N-to y Transcription Products GGGCGAAT-N-UU GGGCGAAT-N-UU w N Deoxyribozyme Cleavage - Q GGGCGAAT -------' Racction GGGCGAAT N-- UU N-UU ssRNA products N-UU Anneal ssRNA UU S-2N- UU siRNA product FIGURE 1: Flowchart summarising the procedure for siRNA synthesis. Patent Application Publication May 6, 2004 Sheet 2 of 2 US 2004/0086860 A1 Full-length transcript 3'-digestion product 5'-digestion product (5'GGGCGAATA) A: Production of single-stranded RNA templates by in vitro transcription and digestion With a deoxyribozyme V 2- 2 V 22inv 22 * 2 &3 S/AS - 88.8x, *...* or as IGFR -- is as 4. -
INTRODUCTION Sirna and Rnai
J Korean Med Sci 2003; 18: 309-18 Copyright The Korean Academy ISSN 1011-8934 of Medical Sciences RNA interference (RNAi) is the sequence-specific gene silencing induced by dou- ble-stranded RNA (dsRNA). Being a highly specific and efficient knockdown tech- nique, RNAi not only provides a powerful tool for functional genomics but also holds Institute of Molecular Biology and Genetics and School of Biological Science, Seoul National a promise for gene therapy. The key player in RNAi is small RNA (~22-nt) termed University, Seoul, Korea siRNA. Small RNAs are involved not only in RNAi but also in basic cellular pro- cesses, such as developmental control and heterochromatin formation. The inter- Received : 19 May 2003 esting biology as well as the remarkable technical value has been drawing wide- Accepted : 23 May 2003 spread attention to this exciting new field. V. Narry Kim, D.Phil. Institute of Molecular Biology and Genetics and School of Biological Science, Seoul National University, San 56-1, Shillim-dong, Gwanak-gu, Seoul 151-742, Korea Key Words : RNA Interference (RNAi); RNA, Small interfering (siRNA); MicroRNAs (miRNA); Small Tel : +82.2-887-8734, Fax : +82.2-875-0907 hairpin RNA (shRNA); mRNA degradation; Translation; Functional genomics; Gene therapy E-mail : [email protected] INTRODUCTION established yet, testing 3-4 candidates are usually sufficient to find effective molecules. Technical expertise accumulated The RNA interference (RNAi) pathway was originally re- in the field of antisense oligonucleotide and ribozyme is now cognized in Caenorhabditis elegans as a response to double- being quickly applied to RNAi, rapidly improving RNAi stranded RNA (dsRNA) leading to sequence-specific gene techniques. -
Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas Reinhardtii
University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations and Theses in Biological Sciences Biological Sciences, School of Spring 2013 Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii Xinrong Ma University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/bioscidiss Part of the Biology Commons, Cellular and Molecular Physiology Commons, Microbiology Commons, and the Molecular Genetics Commons Ma, Xinrong, "Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii" (2013). Dissertations and Theses in Biological Sciences. 51. https://digitalcommons.unl.edu/bioscidiss/51 This Article is brought to you for free and open access by the Biological Sciences, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations and Theses in Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII by Xinrong Ma A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biological Sciences Under the Supervision of Professor Heriberto Cerutti Lincoln, Nebraska May, 2013 SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII Xinrong Ma, Ph.D. University of Nebraska, 2013 Advisor: Heriberto Cerutti RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes, with regulatory roles in a variety of biological processes, including cell cycle, cell differentiation, physiological and metabolic pathways, and stress responses. -
Replication and Kinetic Trapping of Nucleic Acids in Alternative Environments
REPLICATION AND KINETIC TRAPPING OF NUCLEIC ACIDS IN ALTERNATIVE ENVIRONMENTS A Dissertation Presented to The Academic Faculty by Adriana Lozoya Colinas In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Chemistry and Biochemistry Georgia Institute of Technology December 2020 COPYRIGHT © 2020 BY ADRIANA LOZOYA COLINAS REPLICATION AND KINETIC TRAPPING OF NUCLEIC ACIDS IN ALTERNATIVE ENVIRONMENTS Approved by: Dr. Nicholas V. Hud, Advisor Dr. Amanda Stockton School of Chemistry and Biochemistry School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Martha A. Grover Dr. Adegboyega (Yomi) Oyelere School of Chemical & Biomolecular School of Chemistry and Biochemistry Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Loren Williams School of Chemistry and Biochemistry Georgia Institute of Technology Date Approved: October 16, 2020 ACKNOWLEDGEMENTS I would like to thank my mom and dad for all their support and setting up an example for me to follow. I really appreciate everything you have done to encourage me to succeed and follow my dreams. I want to thank Mario for always supporting me. I know it hasn’t always been easy being far away, thank you for being patient and supportive with me. Thank you for the time and adventures we lived together. To all my Latino family at Georgia Tech, thank you for making me feel closer to home. We spent a lot of time together, learned a lot from each other and shared our culture, all of which have made my PhD experience more enjoyable. I would also like to acknowledge my advisor, Nick Hud, for being supportive and sharing his passion for science with me. -
Guide for Morpholino Users: Toward Therapeutics
Open Access Journal of Drug Discovery, Development and Delivery Special Article - Antisense Drug Research and Development Guide for Morpholino Users: Toward Therapeutics Moulton JD* Gene Tools, LLC, USA Abstract *Corresponding author: Moulton JD, Gene Tools, Morpholino oligos are uncharged molecules for blocking sites on RNA. They LLC, 1001 Summerton Way, Philomath, Oregon 97370, are specific, soluble, non-toxic, stable, and effective antisense reagents suitable USA for development as therapeutics and currently in clinical trials. They are very versatile, targeting a wide range of RNA targets for outcomes such as blocking Received: January 28, 2016; Accepted: April 29, 2016; translation, modifying splicing of pre-mRNA, inhibiting miRNA maturation and Published: May 03, 2016 activity, as well as less common biological targets and diagnostic applications. Solutions have been developed for delivery into a range of cultured cells, embryos and adult animals; with development of a non-toxic and effective system for systemic delivery, Morpholinos have potential for broad therapeutic development targeting pathogens and genetic disorders. Keywords: Splicing; Duchenne muscular dystrophy; Phosphorodiamidate morpholino oligos; Internal ribosome entry site; Nonsense-mediated decay Morpholinos: Research Applications, the transcript from miRNA regulation; Therapeutic Promise • Block regulatory proteins from binding to RNA, shifting Morpholino oligos bind to complementary sequences of RNA alternative splicing; and get in the way of processes. Morpholino oligos are commonly • Block association of RNAs with cytoskeletal motor protein used to prevent a particular protein from being made in an organism complexes, preventing RNA translocation; or cell culture. Morpholinos are not the only tool used for this: a protein’s synthesis can be inhibited by altering DNA to make a null • Inhibit poly-A tailing of pre-mRNA; mutant (called a gene knockout) or by interrupting processes on RNA • Trigger frame shifts at slippery sequences; (called a gene knockdown). -
Long Non-Coding Rnas Are Largely Dispensable for Zebrafish Embryogenesis, Viability and Fertility
bioRxiv preprint doi: https://doi.org/10.1101/374702; this version posted July 23, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Long non-coding RNAs are largely dispensable for zebrafish embryogenesis, viability and fertility Mehdi Goudarzi1, Kathryn Berg1, Lindsey M. Pieper1, and Alexander F. Schier1,2,3,4,5 1Department oF Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. 2Center For Brain Science, Harvard University, Cambridge, MA 02138, USA., 3FAS Center For Systems Biology, Harvard University, Cambridge, MA 02138, USA., 4Allen Discovery Center For Cell Lineage Tracing, University oF Washington, Seattle, 5 WA 98195, USA., Biozentrum, University oF Basel, Switzerland. Correspondence should be addressed to M.G. ([email protected]) or A.F.S. ([email protected]). Hundreds oF long non-coding RNAs (lncRNAs) have been identiFied as potential regulators oF gene expression, but their Functions remain largely unknown. To study the role oF lncRNAs during vertebrate development, we selected 25 zebraFish lncRNAs based on their conservation, expression proFile or proximity to developmental regulators, and used CRISPR-Cas9 to generate 32 deletion alleles. We observed altered transcription oF neighboring genes in some mutants, but none oF the lncRNAs were required For embryogenesis, viability or Fertility. Even RNAs with previously proposed non-coding Functions (cyrano and squint) and other conserved lncRNAs (gas5 and lnc-setd1ba) were dispensable. In one case (lnc-phox2bb), absence oF putative DNA regulatory-elements, but not of the lncRNA transcript itselF, resulted in abnormal development. -
Retroviral Delivery of Small Interfering RNA Into Primary Cells
Retroviral delivery of small interfering RNA into primary cells Gregory M. Barton and Ruslan Medzhitov* Section of Immunobiology and The Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520 Communicated by Peter Cresswell, Yale University School of Medicine, New Haven, CT, October 2, 2002 (received for review August 9, 2002) RNA interference is an evolutionarily conserved process in which gene was replaced with the human CD4 gene, which was cloned recognition of double-stranded RNA ultimately leads to posttran- from splenic cDNA. To eliminate any potential signaling, a scriptional suppression of gene expression. This suppression is premature stop codon was introduced after amino acid 425, just mediated by short (21- to 22-nt) small interfering RNAs (siRNAs), after the transmembrane domain. The human H1 promoter was which induce degradation of mRNA based on complementary base cloned from genomic DNA and inserted either upstream of the pairing. The silencing of gene expression by siRNAs is emerging cytomegalovirus (CMV) promoter (RVH1) by using previously rapidly as a powerful method for genetic analysis. Recently, several introduced XhoI and EcoRI sites or within the 3Ј LTR by groups have reported systems designed to express siRNAs in using SalI. The oligonucleotides encoding the human p53 mammalian cells through transfection of either oligonucleotides or siRNA, described by Brummelkamp et al. (6), were 5Ј-GATC- plasmids encoding siRNAs. Because these systems rely on trans- CCCGACTCCAGTGGTAATCTACTTCAAGAGAGTA- fection for delivery, the cell types available for study are restricted GATTACCACTGGAGTCTTTTTGGAAC-3Ј and 5Ј-TCGA- generally to transformed cell lines. Here, we describe a retroviral GTTCCAAAAAGACTCCAGTGGTAATCTACTCTCTTG- system for delivery of siRNA into cells. -
Viral Vectors Applied for Rnai-Based Antiviral Therapy
viruses Review Viral Vectors Applied for RNAi-Based Antiviral Therapy Kenneth Lundstrom PanTherapeutics, CH1095 Lutry, Switzerland; [email protected] Received: 30 July 2020; Accepted: 21 August 2020; Published: 23 August 2020 Abstract: RNA interference (RNAi) provides the means for alternative antiviral therapy. Delivery of RNAi in the form of short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA) have demonstrated efficacy in gene silencing for therapeutic applications against viral diseases. Bioinformatics has played an important role in the design of efficient RNAi sequences targeting various pathogenic viruses. However, stability and delivery of RNAi molecules have presented serious obstacles for reaching therapeutic efficacy. For this reason, RNA modifications and formulation of nanoparticles have proven useful for non-viral delivery of RNAi molecules. On the other hand, utilization of viral vectors and particularly self-replicating RNA virus vectors can be considered as an attractive alternative. In this review, examples of antiviral therapy applying RNAi-based approaches in various animal models will be described. Due to the current coronavirus pandemic, a special emphasis will be dedicated to targeting Coronavirus Disease-19 (COVID-19). Keywords: RNA interference; shRNA; siRNA; miRNA; gene silencing; viral vectors; RNA replicons; COVID-19 1. Introduction Since idoxuridine, the first anti-herpesvirus antiviral drug, reached the market in 1963 more than one hundred antiviral drugs have been formally approved [1]. Despite that, there is a serious need for development of novel, more efficient antiviral therapies, including drugs and vaccines, which has become even more evident all around the world today due to the recent coronavirus pandemic [2]. -
Targeting Protein Translation, RNA Splicing, and Degradation by Morpholino-Based Conjugates in Plasmodium Falciparum
Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum Aprajita Garga, Donna Wesolowskib, Dulce Alonsob,1, Kirk W. Deitschc, Choukri Ben Mamouna, and Sidney Altmanb,2 aDepartment of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; bDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; and cDepartment of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065 Contributed by Sidney Altman, August 11, 2015 (sent for review May 27, 2015; reviewed by Ron Dzikowski and Rima Mcleod) Identification and genetic validation of new targets from available same targets, MO conjugates have also been used to inhibit RNA genome sequences are critical steps toward the development of splicing and initiation of protein translation (9, 17, 18). new potent and selective antimalarials. However, no methods are To enhance cellular uptake of morpholino oligomers and other currently available for large-scale functional analysis of the Plasmo- drug-like molecules, arginine-rich peptides and polyguanidino dium falciparum genome. Here we present evidence for successful dendrimers have been used (19–23). For morpholino-based anti- use of morpholino oligomers (MO) to mediate degradation of target microbial activity, two types of conjugates have been developed, mRNAs or to inhibit RNA splicing or translation of several genes of PPMOs and vivo morpholino oligomers (VMOs, octa-guanidinium P. falciparum involved in chloroquine transport, apicoplast biogen- dendrimer-conjugated MOs; Materials and Methods). PPMOs are esis, and phospholipid biosynthesis. Consistent with their role in the produced following conjugation of a specific MO to a cell-pen- parasite life cycle, down-regulation of these essential genes resulted etrating, arginine-rich peptide, whereas VMOs are synthesized in inhibition of parasite development. -
Advances in Oligonucleotide Drug Delivery
REVIEWS Advances in oligonucleotide drug delivery Thomas C. Roberts 1,2 ✉ , Robert Langer 3 and Matthew J. A. Wood 1,2 ✉ Abstract | Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge. Oligonucleotides are nucleic acid polymers with the In addition to their ability to recognize specific tar- potential to treat or manage a wide range of diseases. get sequences via complementary base pairing, nucleic Although the majority of oligonucleotide therapeutics acids can also interact with proteins through the for- have focused on gene silencing, other strategies are being mation of three-dimensional secondary structures — a pursued, including splice modulation and gene activa- property that is also being exploited therapeutically. For tion, expanding the range of possible targets beyond example, nucleic acid aptamers are structured