EP0473576A1 - SHORT THERAPEUTIC dsRNA OF DEFINED STRUCTURE - Google Patents

SHORT THERAPEUTIC dsRNA OF DEFINED STRUCTURE

Info

Publication number
EP0473576A1
EP0473576A1 EP89906635A EP89906635A EP0473576A1 EP 0473576 A1 EP0473576 A1 EP 0473576A1 EP 89906635 A EP89906635 A EP 89906635A EP 89906635 A EP89906635 A EP 89906635A EP 0473576 A1 EP0473576 A1 EP 0473576A1
Authority
EP
European Patent Office
Prior art keywords
dsrna
human
therapeutically
short
therapy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP89906635A
Other languages
German (de)
French (fr)
Other versions
EP0473576A4 (en
Inventor
David H. Gillespie
William A. Carter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hem Research Inc
Original Assignee
Hem Research Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hem Research Inc filed Critical Hem Research Inc
Publication of EP0473576A1 publication Critical patent/EP0473576A1/en
Publication of EP0473576A4 publication Critical patent/EP0473576A4/en
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention generally relates to therapeutic compositions of matter, methods for producing said compositions and methods for administering said compositions to living organisms, including human beings.
  • dsRNA long, double-stranded RNA
  • dsRNA poly(I) :poly(C) and poly(I):poly (C 12 ,U)
  • Ampligen® are anticancer and antiAIDS agents (1).
  • These dsRNAs induce interferon and activate a variety of cellular enzymes (2).
  • These dsRNAs are enzymatically synthesized as high molecular weight nucleic acid polymers (m>300), using ribonucleoside diphosphates as substrate and polynucleotide phosphorylase (PNPase) as enzyme. Ampligen was created because its parent compound, poly(I) :poly(C), was toxic (3). In the 1960's, Drs.
  • Such short dsRNAs having the proper nucleotide sequence will be therapeutic by virtue of their biological activity. It should be emphasized that the prior art teaches away from the present invention by teaching the need for long, biodegradable dsRNA in order to preserve biological activity without toxicity.
  • Short dsRNA of defined sequence cannot be synthesized by the PNPase method.
  • PNPase cannot synthesize a nucleic acid of defined sequence because it is a terminal transferase and not a template-copying enzyme.
  • the helical content of dsRNAs of the homopolymer:homopolymer type constantly changes due to the "slippage" reaction. Slippage means that the two strands of the dsRNA molecule mover relative to each other since there is no complementary nucleotide register to fix the position of two strands relative to each other.
  • the present invention produces means for synthesizing stable short dsRNA of defined sequence.
  • FIG. 1 is an illustration of two types of short dsRNA of defined sequence.
  • a dsRNA with terminal "locks” while in FIG. IB is shown a dsRNA with a central "hinge”.
  • the dsRNA in FIG. 1 also contain "internal registers", which are indicated by A-U base pairs.
  • FIG. 2 is an illustration of a method for preparing a short dsRNA of defined sequence having terminal locks and internal registers.
  • FIG. 3 is an illustration f a method for preparing short dsRNA of defined sequence having a central or near-central hinge.
  • FIG. 4 is an illustration of a method for preparing short dsRNA of defined sequence having both terminal locks and a central or near-central hinge.
  • Types of dsRNA Types of dsRNA.
  • the present compositions of matter and methods relative to their use generally depend on the several embodiments thereof on the chemical modifications of protein-inducing and/or enzyme-activating dsRNA complex to render said complex less toxic to a living animal cell.
  • the chemically modified complexes disclosed herein retain the biological activity of unmodified complexes while being less toxic by mechanisms which are presently mysterious.
  • the dsRNA complexes which are of concern in the present invention may be modified by shortening said dsRNA while at the same time fixing the two strands in space relative to one another.
  • Figure 1 depicts two types of dsRNA envisioned in this application. Type A dsRNA is termed "locked dsRNA".
  • Hinged dsRNA contains an internal self-complementary stretch which folds in a restricted way to align the remaining dsRNA nucleotides (see also Figures 3 and 4 for example of hinged dsRNA) .
  • locks are complementary nucleotide pairs different from homopolymer stretches in the dsRNA.
  • Said locks, hinges and internal registers are referred to as heteropolymer regions in this application.
  • At one extreme is a homopolymer with a single heteropolymer nucleotide pair to fix the dsRNA register while at the other extreme is a totally heteropolymeric short dsRNA.
  • Both locked and hinged dsRNAs may contain single stranded regions terminally or internally ( Figure 2).
  • Locked RNA molecules of defined length and defined sequence can be synthesized from plasmid DNA vectors having promoters of defined sequence placed near the sequence of interest.
  • the vectors, enzymes and substrates are available from a variety of commercial sources.
  • the locked dsRNA depicted as structure [7] of Figure 2 can be made as follows.
  • the two deoxyoligonucleotides depicted at the top of Figure 2 (structure [1]) can be synthesized by an oligonucleotide synthesizer.
  • Hinged RNA molecules of defined length and defined sequence can be synthesized from plasmid DNA vectors having promoters of defined sequence placed near the sequence of interest.
  • the hinged dsRNA depicted as structure [8] of Figure 3 can be made as follows: The two deoxyoligunucleotides depicted at the top of Figure 3 (structures!II ) can be synthesized by an oligunucleotide synthesizer, annealed to produce structure [2] and cloned into pGEM4 previously cut with Eco Rl and Sma 1 yielding structure [3] .
  • RNA structure [4] which can be self-annealed to produce the dsRNA shown as structure [5] .
  • This hinged dsRNA can be used as is or can be trimmed with RNase to produce the dsRNA shown as structure ([6]), Figure 3.
  • dsRNA molecules of defined length and sequence with both locks and hinges can be synthesized by a slight modification of this above procedure, using Hind II instead of Sma 1 and using slightly different deoxyoligonucleotides, as depicted in Figure 4.
  • oligonucleotides conisting of RNA polymerase promoters flanking inserts specifying the present invention can be synthesized, annealed and transcribed directly, without cloning into a vector.
  • short dsRNA can be chemically synthesized. The essence of this invention lies in the structure and properties of the dsRNAs themselves; this example is given to enable one with ordinary skill in the art to prepare short therapeutic dsRNAs of defined sequence.
  • dsRNA Determining Biological Activity of Short dsRNA of Defined Sequence.
  • the biological activity of dsRNA can be assessed in several experimental systems which are routine in the art.
  • the antiviral properties of dsRNA can be measured by challenging dsRNA-treated cells with vesicular stomatitis virus (VSV) and measuring reduction in virus yield as described by (5). Similar procedures have been reported which measure the inhibition of VSV and other viruses.
  • VSV vesicular stomatitis virus
  • the antitumor properties of dsRNA can be evaluated by exposing tumor cells in tissue culture to dsRNA and measuring reduction in growth rate as described by (6).
  • the antitumor properties of dsRNA can also be measured by injecting dsRNA into nude mice bearing tumors and measuring tumor growth rate (7).
  • dsRNA to enhance natural killer cell or macrophage killing activity
  • All of these procedures are routine in the art and are cited by way of enabling one with ordinary skill in the art to measure the biological activity of dsRNA synthesized as described in the preceding section. The citing of these procedures should not be construed as limiting; other procedures for measuring the biological activity of dsRNA exist and are also well known in the art. 4) Determining the Absence of Toxicity of dsRNA of Defined Sequence.
  • dsRNA The toxicity or lack of it of dsRNA can be determined by procedures which have long been routine for testing a variety of potential therapeutics and which have long been routine for testing a variety of potential therapeutics and which have been applied to dsRNA as well.
  • Suitable test animals such as mice, rats, rabbits, dogs, monkeys, etc. or humans can be injected periodically with various quantities of dsRNA and after a suitable interval, such animals can be examined for evidence of fever, loss of weight, loss of liver function, thrombocytopenia, leukopenia, bone marrow suppression, etc.
  • the examiner is directed particularly to Citations (9) for examples where such studies have been done regarding dsRNA.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne l'induction de protéines ainsi que l'activation d'enzymes dans les cellules d'organismes vivants, parmi lesquels l'homme. Selon l'invention on modifie des complexes d'acides nucléiques longs, tels que des complexes de polyriboinosinate et de polycytidylate, afin de produire un ARN bicaténaire court de séquence définie. Lesdits ARN bicaténaires courts de séquence définie conservent la capacité d'induire des protéines et d'activer des enzymes, mais ne sont pas aussi toxiques que les ARN bicaténaires longs.The invention relates to the induction of proteins as well as the activation of enzymes in the cells of living organisms, including humans. According to the invention, complexes of long nucleic acids, such as polyriboinosinate and polycytidylate complexes, are modified in order to produce a short double-stranded RNA of defined sequence. Said short double-stranded RNAs of defined sequence retain the ability to induce proteins and activate enzymes, but are not as toxic as long double-stranded RNAs.

Description

SHORT THERAPEUTIC dsRNA OF DEFINED STRUCTURE
BACKGROUND AND SUMMARY
The invention generally relates to therapeutic compositions of matter, methods for producing said compositions and methods for administering said compositions to living organisms, including human beings.
Certain long, double-stranded RNA (dsRNA), especially poly(I) :poly(C) and poly(I):poly (C12,U) (Ampligen®) are anticancer and antiAIDS agents (1). These dsRNAs induce interferon and activate a variety of cellular enzymes (2). These dsRNAs are enzymatically synthesized as high molecular weight nucleic acid polymers (m>300), using ribonucleoside diphosphates as substrate and polynucleotide phosphorylase (PNPase) as enzyme. Ampligen was created because its parent compound, poly(I) :poly(C), was toxic (3). In the 1960's, Drs. Carter and Ts'o reasoned that a metabolically unstable, long dsRNA derivative might be cleared quickly from blood and therefore exhibit minimal toxicity, (4). They created Ampligen® as a long dsRNA molecule with RNase-sensitive mismatches an this molecule retained biologic potence while proving to be non-toxic (1). To indicate the size of poly(I) :poly(C) or poly(I):poly (C_.7,U) in general use, the inventors point out that the specifications for Ampligen in clinical trials in 198701988 include a requirement of an S20, w of 10-15, corresponding to a molecular weight in excess of 1,000,000 and a number of base pairs in excess of 1500.
A new line of thinking has lead the present inventors to a new and different mechanism account for biological activity and lack of toxicity simultaneously characterizing dsRNA, allowing the inventors to create a new class of dsRNA molecule with therapeutic benefit. In contrast to the teachings of the prior art, the inventors have concluded that lack of toxicity of Ampligen derives from the helical interruptions (i.e., shortness of helical stretches) introduced by uracil residues and not from RNase sensitivity. Thus, the essence of this invention is as follows: First, certain short dsRNAs will be nontoxic and biologically active, regardless of whether they contain RNAse-sensitive mismatches. Such short dsRNAs having the proper nucleotide sequence will be therapeutic by virtue of their biological activity. It should be emphasized that the prior art teaches away from the present invention by teaching the need for long, biodegradable dsRNA in order to preserve biological activity without toxicity.
Short dsRNA of defined sequence cannot be synthesized by the PNPase method. First, PNPase cannot synthesize a nucleic acid of defined sequence because it is a terminal transferase and not a template-copying enzyme. Second, the helical content of dsRNAs of the homopolymer:homopolymer type constantly changes due to the "slippage" reaction. Slippage means that the two strands of the dsRNA molecule mover relative to each other since there is no complementary nucleotide register to fix the position of two strands relative to each other. The present invention produces means for synthesizing stable short dsRNA of defined sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration of two types of short dsRNA of defined sequence. In FIG. 1A is shown a dsRNA with terminal "locks" while in FIG. IB is shown a dsRNA with a central "hinge". The dsRNA in FIG. 1 also contain "internal registers", which are indicated by A-U base pairs.
FIG. 2 is an illustration of a method for preparing a short dsRNA of defined sequence having terminal locks and internal registers.
FIG. 3 is an illustration f a method for preparing short dsRNA of defined sequence having a central or near-central hinge.
FIG. 4 is an illustration of a method for preparing short dsRNA of defined sequence having both terminal locks and a central or near-central hinge.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
1) Types of dsRNA. The present compositions of matter and methods relative to their use generally depend on the several embodiments thereof on the chemical modifications of protein-inducing and/or enzyme-activating dsRNA complex to render said complex less toxic to a living animal cell. The chemically modified complexes disclosed herein retain the biological activity of unmodified complexes while being less toxic by mechanisms which are presently mysterious. The dsRNA complexes which are of concern in the present invention may be modified by shortening said dsRNA while at the same time fixing the two strands in space relative to one another. Figure 1 depicts two types of dsRNA envisioned in this application. Type A dsRNA is termed "locked dsRNA". It contains complementary regions at each end ("locks") to fix the dsRNA register. Variants of this model may contain subterminal, rather than or in addition to, terminal locks or may contain only one lock. Locks may be as short as a single nucleotide. In addition, "internal registers" like the A-U base pairs of Figures 1 and 2 may be added for increased stability. Type B dsRNA is termed "hinged" dsRNA. Hinged dsRNA contains an internal self-complementary stretch which folds in a restricted way to align the remaining dsRNA nucleotides (see also Figures 3 and 4 for example of hinged dsRNA) . For the purposes of this application "locks", "hinges" and "internal registers" are complementary nucleotide pairs different from homopolymer stretches in the dsRNA. Said locks, hinges and internal registers are referred to as heteropolymer regions in this application. At one extreme is a homopolymer with a single heteropolymer nucleotide pair to fix the dsRNA register while at the other extreme is a totally heteropolymeric short dsRNA. Both locked and hinged dsRNAs may contain single stranded regions terminally or internally (Figure 2).
2) Synthesis of Short dsRNAs of Defined Sequence. Locked RNA molecules of defined length and defined sequence can be synthesized from plasmid DNA vectors having promoters of defined sequence placed near the sequence of interest. The vectors, enzymes and substrates are available from a variety of commercial sources. For example the locked dsRNA depicted as structure [7] of Figure 2 can be made as follows. The two deoxyoligonucleotides depicted at the top of Figure 2 (structure [1]) can be synthesized by an oligonucleotide synthesizer. Annealing them as shown (structure [2]) leaves single-stranded ends which can be cloned into the commercially available vector, pGEM 4, after cleavage of the vector with EcoRl and Hind III, yielding structure [3]. Transcription of this plasmid as described in Figure 2 yields separately two single stranded RNAs (structures [4] and [5]) which can be annealed (e.g., at 65° in 1M NaP04, pH7) to produce the dsRNA shown in structure [6] in Figure 2. This locked dsRNA can be used as is or can be trimmed with RNAse to produce the dsRNA shown in structure [7] , Figure 2.
Hinged RNA molecules of defined length and defined sequence can be synthesized from plasmid DNA vectors having promoters of defined sequence placed near the sequence of interest. For example, the hinged dsRNA depicted as structure [8] of Figure 3 can be made as follows: The two deoxyoligunucleotides depicted at the top of Figure 3 (structures!II ) can be synthesized by an oligunucleotide synthesizer, annealed to produce structure [2] and cloned into pGEM4 previously cut with Eco Rl and Sma 1 yielding structure [3] . Transcription of this plasmid as described in Figure 3 yields an RNA (structure [4] which can be self-annealed to produce the dsRNA shown as structure [5] . This hinged dsRNA can be used as is or can be trimmed with RNase to produce the dsRNA shown as structure ([6]), Figure 3. dsRNA molecules of defined length and sequence with both locks and hinges can be synthesized by a slight modification of this above procedure, using Hind II instead of Sma 1 and using slightly different deoxyoligonucleotides, as depicted in Figure 4.
It will be obvious to those with ordinary skill in the art that other vectors and other restriction endonuclease sites can be used with similar results.
It will also be obvious that other degrees of repetition than 3 of [ (I10)A]/[ (C-,0)U] can be employed and that other polypurine/polypyrimidine tracts can be used, such as ( ^r_)/(.C r_)' (I)n/[(C);U[ ,
(A)m/(U)m, etc., so long as helical regions are kept short enough to avoid toxicity and long enough to retain biological activity.
It will also be obvious to those with ordinary skill in the art that oligonucleotides conisting of RNA polymerase promoters flanking inserts specifying the present invention can be synthesized, annealed and transcribed directly, without cloning into a vector. It will also be obvious to those with ordinary skill in the art that short dsRNA can be chemically synthesized. The essence of this invention lies in the structure and properties of the dsRNAs themselves; this example is given to enable one with ordinary skill in the art to prepare short therapeutic dsRNAs of defined sequence.
3) Determining Biological Activity of Short dsRNA of Defined Sequence. The biological activity of dsRNA can be assessed in several experimental systems which are routine in the art. The antiviral properties of dsRNA can be measured by challenging dsRNA-treated cells with vesicular stomatitis virus (VSV) and measuring reduction in virus yield as described by (5). Similar procedures have been reported which measure the inhibition of VSV and other viruses. The antitumor properties of dsRNA can be evaluated by exposing tumor cells in tissue culture to dsRNA and measuring reduction in growth rate as described by (6). The antitumor properties of dsRNA can also be measured by injecting dsRNA into nude mice bearing tumors and measuring tumor growth rate (7). The ability of dsRNA to enhance natural killer cell or macrophage killing activity can be determined as detailed (8). All of these procedures are routine in the art and are cited by way of enabling one with ordinary skill in the art to measure the biological activity of dsRNA synthesized as described in the preceding section. The citing of these procedures should not be construed as limiting; other procedures for measuring the biological activity of dsRNA exist and are also well known in the art. 4) Determining the Absence of Toxicity of dsRNA of Defined Sequence. The toxicity or lack of it of dsRNA can be determined by procedures which have long been routine for testing a variety of potential therapeutics and which have long been routine for testing a variety of potential therapeutics and which have been applied to dsRNA as well. Suitable test animals such as mice, rats, rabbits, dogs, monkeys, etc. or humans can be injected periodically with various quantities of dsRNA and after a suitable interval, such animals can be examined for evidence of fever, loss of weight, loss of liver function, thrombocytopenia, leukopenia, bone marrow suppression, etc. The examiner is directed particularly to Citations (9) for examples where such studies have been done regarding dsRNA.

Claims

WHAT IS CLAIMED IS:
1. Short dsRNA of defined structure, said dsRNA having biological activity and lacking significant toxicity.
2. The dsRNA of Claim 1 stabilized by "lock", "hinge" and/or "internal register" regions of complementary heteropolymer.
3. The dsRNA of Claim 2 having the general structure:
where said "m" and said "n" are less than 100 and more than 5 and where said locks in one strand are complementary to locks in the opposite strand and where said "I" and said "C" are inosine monophosphate and cytidine monophosphate, respectively.
4. The dsRNA of Claim 2 having the general structure 5' (I)n-hinge-(C)m3', where said "m" and said "n" are less than 100 and more than 5 and where said hinge is a heteropolymeric region exhibiting self-complementarity and where said "I" and said "C" are inosine monophosphate and cytidine monophosphate respectively.
5. The dsRNAs of any one of Claims 1-5 having the general structure 3 5 ' lock- [ ( I ) A] . -lock 3 *
4 ^ 3 ' lock- [ (C) ' UK k-lock 3 '
5 where said "m" and said "n" are less than 25 and more
6 than 5, where said "j" and said "k" are less than 10
7 and more than 0, where said "I" and said "C" are
8 inosine monophosphate and cytidine monophosphate,
9 respectively, where said "A" is a nucleotide which is
10 not I and where "U" is a nucleotide which base pairs
11 with said A.
1 7. The dsRNA of Claim 1 with substitutions in one
2 strand, said substitutions being not complementary to'
3 nucleotides in the opposite strand.
1 8. The dsRNA of Claim 7, said dsRNAs having
2 single-stranded tails.
1 9. A method of therapeutically activating
2 dsRNA-dependent enzymes or inducing interferon in a
3 human in need of such therapy, which method comprises administering to the human a therapeutically
5 effective amount of the short dsRNA of defined ø structure according to claim 1.
1 10. A method of therapeutically activating
2 dsRNA-dependent enzymes or inducing interferon in a
3 human in need of such therapy, which method comprises
4 administering to the human a therapeutically
5 effective amount of the short dsRNA of defined
6 structure according to claim 2.
1 11. A method of therapeutically activating dsRNA dependent enzymes or inducing interferon in a human in need of such therapy, which method comprises administering to the human a therapeutically effective amount of the short dsRNA of defined structure according to claim 3.
12. A method of therapeutically activating dsRNA dependent enzymes or inducing interferon in a human in need of such therapy, which method comprises administering to the human a therapeutically effective amount of the short dsRNA of defined structure according to claim 4.
13. A method of therapeutically activating dsRNA dependent enzymes or inducing interferon in a human in need of such therapy, which method comprises administering to the human a therapeutically effective amount of the short dsRNA of defined structure according to claim 5.
14. A method of therapeutically activating dsRNA dependent enzymes or inducing interferon in a human in need of such therapy, which method comprises administering to the human a therapeutically effective amount of the short dsRNA of defined structure according to claim 6.
15. A method of therapeutically activating dsRNA dependent enzymes or inducing interferon in a human in need of such therapy, which method comprises administering to the human a therapeutically effective amount of the short dsRNA of defined structure according to claim 7.
16. A method of therapeutically activating dsRNA dependent enzymes or inducing interferon in a human in need of such therapy, which method comprises administering to the human a therapeutically effective amount of the short dsRNA of defined structure according to claim 8.
EP19890906635 1989-05-19 1989-05-19 Short therapeutic dsrna of defined structure Ceased EP0473576A4 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1989/002172 WO1990014090A1 (en) 1989-05-19 1989-05-19 SHORT THERAPEUTIC dsRNA OF DEFINED STRUCTURE

Publications (2)

Publication Number Publication Date
EP0473576A1 true EP0473576A1 (en) 1992-03-11
EP0473576A4 EP0473576A4 (en) 1993-03-10

Family

ID=22215026

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19890906635 Ceased EP0473576A4 (en) 1989-05-19 1989-05-19 Short therapeutic dsrna of defined structure

Country Status (5)

Country Link
EP (1) EP0473576A4 (en)
JP (1) JPH04507083A (en)
DK (1) DK188991D0 (en)
NO (1) NO914529L (en)
WO (1) WO1990014090A1 (en)

Families Citing this family (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2685346B1 (en) * 1991-12-18 1994-02-11 Cis Bio International PROCESS FOR THE PREPARATION OF DOUBLE-STRANDED RNA, AND ITS APPLICATIONS.
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
CA2513336A1 (en) 1998-03-20 1999-09-30 Benitec Australia Ltd. Control of gene expression in a non-human eukaryotic cell, tissue or organ
AUPP249298A0 (en) 1998-03-20 1998-04-23 Ag-Gene Australia Limited Synthetic genes and genetic constructs comprising same I
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
US20040214330A1 (en) 1999-04-07 2004-10-28 Waterhouse Peter Michael Methods and means for obtaining modified phenotypes
GB9827152D0 (en) * 1998-07-03 1999-02-03 Devgen Nv Characterisation of gene function using double stranded rna inhibition
US7005423B1 (en) 1999-07-02 2006-02-28 Devgen Nv Characterization of gene function using double stranded RNA inhibition
AU776150B2 (en) 1999-01-28 2004-08-26 Medical College Of Georgia Research Institute, Inc. Composition and method for (in vivo) and (in vitro) attenuation of gene expression using double stranded RNA
AU4077100A (en) 1999-04-08 2000-11-14 Chiron Corporation Enhancement of the immune response for vaccine and gene therapy applications
BR0010496A (en) * 1999-05-10 2002-04-02 Syngenta Participations Ag Regulation of viral gene expression
US6423885B1 (en) 1999-08-13 2002-07-23 Commonwealth Scientific And Industrial Research Organization (Csiro) Methods for obtaining modified phenotypes in plant cells
US7833992B2 (en) 2001-05-18 2010-11-16 Merck Sharpe & Dohme Conjugates and compositions for cellular delivery
US8273866B2 (en) 2002-02-20 2012-09-25 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
US8202979B2 (en) 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
US7491805B2 (en) 2001-05-18 2009-02-17 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
WO2003070918A2 (en) * 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Rna interference by modified short interfering nucleic acid
WO2002081628A2 (en) 2001-04-05 2002-10-17 Ribozyme Pharmaceuticals, Incorporated Modulation of gene expression associated with inflammation proliferation and neurite outgrowth, using nucleic acid based technologies
US20080032942A1 (en) 2000-08-30 2008-02-07 Mcswiggen James RNA interference mediated treatment of Alzheimer's disease using short interfering nucleic acid (siNA)
EP1229134A3 (en) 2001-01-31 2004-01-28 Nucleonics, Inc Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell
US20050159378A1 (en) 2001-05-18 2005-07-21 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Myc and/or Myb gene expression using short interfering nucleic acid (siNA)
US20050014172A1 (en) 2002-02-20 2005-01-20 Ivan Richards RNA interference mediated inhibition of muscarinic cholinergic receptor gene expression using short interfering nucleic acid (siNA)
US7109165B2 (en) 2001-05-18 2006-09-19 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US9994853B2 (en) 2001-05-18 2018-06-12 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
US20050256068A1 (en) 2001-05-18 2005-11-17 Sirna Therapeutics, Inc. RNA interference mediated inhibition of stearoyl-CoA desaturase (SCD) gene expression using short interfering nucleic acid (siNA)
WO2005078097A2 (en) 2004-02-10 2005-08-25 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING MULTIFUNCTIONAL SHORT INTERFERING NUCLEIC ACID (Multifunctional siNA)
US20050148530A1 (en) 2002-02-20 2005-07-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US8008472B2 (en) 2001-05-29 2011-08-30 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of human immunodeficiency virus (HIV) gene expression using short interfering nucleic acid (siNA)
US7662952B2 (en) 2002-02-20 2010-02-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of GRB2 associated binding protein (GAB2) gene expression using short interfering nucleic acid (siNA)
US8258288B2 (en) 2002-02-20 2012-09-04 Sirna Therapeutics, Inc. RNA interference mediated inhibition of respiratory syncytial virus (RSV) expression using short interfering nucleic acid (siNA)
US20090253774A1 (en) 2002-02-20 2009-10-08 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF PLATELET DERIVED GROWTH FACTOR (PDGF) AND PLATELET DERIVED GROWTH FACTOR RECEPTOR (PDGFR) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7928220B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of stromal cell-derived factor-1 (SDF-1) gene expression using short interfering nucleic acid (siNA)
US7897752B2 (en) 2002-02-20 2011-03-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of telomerase gene expression using short interfering nucleic acid (siNA)
US7910724B2 (en) 2002-02-20 2011-03-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Fos gene expression using short interfering nucleic acid (siNA)
US7897757B2 (en) 2002-02-20 2011-03-01 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of protein tyrosine phosphatase-1B (PTP-1B) gene expression using short interfering nucleic acid (siNA)
US7691999B2 (en) 2002-02-20 2010-04-06 Sirna Therapeutics, Inc. RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering nucleic acid (siNA)
US8013143B2 (en) 2002-02-20 2011-09-06 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of CXCR4 gene expression using short interfering nucleic acid (siNA)
US7667030B2 (en) 2002-02-20 2010-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of matrix metalloproteinase 13 (MMP13) gene expression using short interfering nucleic acid (siNA)
US7700760B2 (en) 2002-02-20 2010-04-20 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular cell adhesion molecule (VCAM) gene expression using short interfering nucleic acid (siNA)
US7683166B2 (en) 2002-02-20 2010-03-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)
US7683165B2 (en) 2002-02-20 2010-03-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)
US7928218B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of polycomb group protein EZH2 gene expression using short interfering nucleic acid (siNA)
US7795422B2 (en) 2002-02-20 2010-09-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hypoxia inducible factor 1 (HIF1) gene expression using short interfering nucleic acid (siNA)
US7935812B2 (en) 2002-02-20 2011-05-03 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of hepatitis C virus (HCV) expression using short interfering nucleic acid (siNA)
US7897753B2 (en) 2002-02-20 2011-03-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of XIAP gene expression using short interfering nucleic acid (siNA)
US7667029B2 (en) 2002-02-20 2010-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of checkpoint kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
US7928219B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (SINA)
US7678897B2 (en) 2002-02-20 2010-03-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
US20090099117A1 (en) 2002-02-20 2009-04-16 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF MYOSTATIN GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US8067575B2 (en) 2002-02-20 2011-11-29 Merck, Sharp & Dohme Corp. RNA interference mediated inhibition of cyclin D1 gene expression using short interfering nucleic acid (siNA)
US20090192105A1 (en) 2002-02-20 2009-07-30 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF INTERCELLULAR ADHESION MOLECULE (ICAM) GENE EXPRESSION USING SHORT INTERFERING NUCELIC ACID (siNA)
US7893248B2 (en) 2002-02-20 2011-02-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Myc and/or Myb gene expression using short interfering nucleic acid (siNA)
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
GB2397062B (en) 2002-02-20 2005-06-15 Sirna Therapeutics Inc RNA interference mediated inhibition of hepatitis c virus (HCV) gene expression using short interfering nucleic acid (siNA)
EP1432724A4 (en) * 2002-02-20 2006-02-01 Sirna Therapeutics Inc Rna interference mediated inhibition of map kinase genes
US20090253773A1 (en) 2002-02-20 2009-10-08 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF TNF AND TNF RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20080274989A1 (en) 2002-08-05 2008-11-06 University Of Iowa Research Foundation Rna Interference Suppression of Neurodegenerative Diseases and Methods of Use Thereof
US20050042646A1 (en) 2002-08-05 2005-02-24 Davidson Beverly L. RNA interference suppresion of neurodegenerative diseases and methods of use thereof
US20040241854A1 (en) 2002-08-05 2004-12-02 Davidson Beverly L. siRNA-mediated gene silencing
US7923547B2 (en) 2002-09-05 2011-04-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10508277B2 (en) 2004-05-24 2019-12-17 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
NL1026335C2 (en) * 2004-06-04 2005-12-06 Univ Delft Tech Method for making a double-stranded polyribonucleotide sequence with overhanging end, as well as a method for forming a double-stranded polynucleotide construct and an application.
CA2584960A1 (en) 2004-10-21 2006-05-04 Charles L. Niblett Methods and materials for conferring resistance to pests and pathogens of plants
ES2663009T3 (en) 2010-10-29 2018-04-10 Sirna Therapeutics, Inc. Inhibition of RNA-mediated gene expression using short interference nucleic acids (ANic)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0300680A2 (en) * 1987-07-17 1989-01-25 Hem Pharmaceuticals Corp. Doublestranded RNA correction of abnormalities in circulating immune complexes and monocyte function
EP0303516A2 (en) * 1987-08-12 1989-02-15 Hem Pharmaceuticals Corp. Topically active compositions of double-stranded RNAs
EP0306347A2 (en) * 1987-09-04 1989-03-08 Hem Pharmaceuticals Corp. Diagnosis of double-stranded RNA deficiency states
EP0308066A2 (en) * 1987-08-12 1989-03-22 Hem Pharmaceuticals Corp. Promotion of host defense by systemic dsRNA treatment
EP0318281A2 (en) * 1987-11-25 1989-05-31 Hem Pharmaceuticals Corp. Use of dsRNA in the prevention of viral escape

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3444043A (en) * 1966-09-30 1969-05-13 Univ Illinois Interference with biologically active rna formation
BR6801650D0 (en) * 1968-08-20 1973-03-07 R Maes PROCESS FOR PREPARING INTERFERON GENERATOR COMPLEXES
SU425940A1 (en) * 1968-09-17 1974-04-30 Н. С. Сидорова, С. Е. Бреслер, Н. А. Зейтленок , Л. М. Вильнер METHOD FOR OBTAINING A SYNTHETIC-WATERPRESSED POLYRIBONUCLEOTIDE COMPLEX
DE1963998A1 (en) * 1968-12-24 1970-11-12 Takeda Chemical Industries Ltd Interferon inducing agent, process for its preparation and methods for inducing interferon by using this agent
US4124702A (en) * 1971-07-06 1978-11-07 Merck & Co., Inc. Polynucleotides active as inducers of interferon production in living animal cells
US3819482A (en) * 1972-05-08 1974-06-25 J Semancik Method of preparing high yields of double-stranded ribonucleic acid
US4024222A (en) * 1973-10-30 1977-05-17 The Johns Hopkins University Nucleic acid complexes
US4024241A (en) * 1974-09-27 1977-05-17 The United States Of America As Represented By The Secretary Of Health, Education And Welfare Nuclease-resistant hydrophilic complex of polyriboinosinic-polyribocytidylic acid
US4018916A (en) * 1975-01-27 1977-04-19 Beecham Group Limited Antiviral ionic complexes of double-standard RNA
JPS5519239A (en) * 1978-07-28 1980-02-09 Green Cross Corp:The Interferon-producing attractant
US4262090A (en) * 1979-06-04 1981-04-14 Cetus Corporation Interferon production
US4349538A (en) * 1979-12-07 1982-09-14 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Nuclease-resistant hydrophilic complex of polyriboinosinic-polyribocytidylic acid
US4388306A (en) * 1980-06-04 1983-06-14 Merck & Co., Inc. Pharmaceutical composition comprising modified polyriboinosinic-polyribocytidylic acid, for induction of interferon in primates
US4400375A (en) * 1981-11-23 1983-08-23 Eli Lilly And Company Tobramycin-double stranded RNA complex suitable for inducing interferon
ATE41159T1 (en) * 1985-08-29 1989-03-15 Berol Kemi Ab CARRIER WITH AN IMMOBILIZED BIOLOGICALLY ACTIVE SUBSTANCE, METHOD OF MANUFACTURE AND ITS USE.

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0300680A2 (en) * 1987-07-17 1989-01-25 Hem Pharmaceuticals Corp. Doublestranded RNA correction of abnormalities in circulating immune complexes and monocyte function
EP0303516A2 (en) * 1987-08-12 1989-02-15 Hem Pharmaceuticals Corp. Topically active compositions of double-stranded RNAs
EP0308066A2 (en) * 1987-08-12 1989-03-22 Hem Pharmaceuticals Corp. Promotion of host defense by systemic dsRNA treatment
EP0306347A2 (en) * 1987-09-04 1989-03-08 Hem Pharmaceuticals Corp. Diagnosis of double-stranded RNA deficiency states
EP0318281A2 (en) * 1987-11-25 1989-05-31 Hem Pharmaceuticals Corp. Use of dsRNA in the prevention of viral escape

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9014090A1 *

Also Published As

Publication number Publication date
DK188991D0 (en) 1991-11-19
EP0473576A4 (en) 1993-03-10
NO914529D0 (en) 1991-11-19
NO914529L (en) 1992-04-14
JPH04507083A (en) 1992-12-10
WO1990014090A1 (en) 1990-11-29

Similar Documents

Publication Publication Date Title
EP0473576A1 (en) SHORT THERAPEUTIC dsRNA OF DEFINED STRUCTURE
DE60038495T2 (en) Oligonucleotide N3'-P5 'thiophosphoramide: its synthesis and use
CN102307997B (en) By suppressing to treat the related disease of Sirtuin 1 (SIRT1) for the natural antisense transcript of Sirtuin 1
RU2523596C2 (en) Single-stranded circular rna and method for producing it
EP0934331B1 (en) Mirror-symmetrical selection and evolution of nucleic acids
DE60207601T2 (en) METHOD FOR THE IN VITRO SYNTHESIS OF SHORT DOBBELSTRÄNGIGEN RNAS
JP5540312B2 (en) Circular single-stranded nucleic acid complex and method for producing the same
JP2003511016A (en) Design of High Affinity RNase H to Replenish Oligonucleotides
Häner et al. Development of artificial ribonucleases
Yamasaki et al. Effect of N-2-acetylaminofluorene modification on the structure and template activity of DNA and reconstituted chromatin
JP2011511776A (en) Cationic siRNA for RNA interference, synthesis and use
US8247540B2 (en) Caged nucleotides and oligonucleotides and their application
JP5252622B2 (en) Double-stranded RNA capable of expressing high nuclease resistance and excellent RNA interference effect
WO2007058323A1 (en) Method for production of nucleic acid homopolymer-bound functional nucleic acid medicine
CN111378659A (en) Nucleic acid for inhibiting STAT3 gene expression, pharmaceutical composition containing nucleic acid and application of pharmaceutical composition
CN114369130A (en) Modified thio-oligonucleotides and uses thereof
WO2020181130A2 (en) Polynucleotide conjugates and uses thereof
Katagiri et al. Artificial oligonucleotides consisting of an analog of nucleoside antibiotics, carbocyclic oxetanocins
CN111154755B (en) Double-stranded oligonucleotide DNA and application thereof
Noronha Synthesis Physicochemical and Biological Studies on Oligonucleotides Containing D-arabinose
EP4019088A1 (en) Rna action inhibitor and use thereof
WO2023192384A1 (en) Tetrazine-derived linkers for single guide rnas
WO2024227047A2 (en) Modified guide rna
JPH01238597A (en) Nucleic acid derivative
KR20240072872A (en) Anticancer Composition Containing Nanoparticle-tRH Conjugate

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19911205

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

A4 Supplementary search report drawn up and despatched

Effective date: 19930115

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17Q First examination report despatched

Effective date: 19950221

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 19970629