WO1996039154A1 - Oligonucleotides having phosphorothioate linkages of high chiral purity - Google Patents

Oligonucleotides having phosphorothioate linkages of high chiral purity Download PDF

Info

Publication number
WO1996039154A1
WO1996039154A1 PCT/US1996/008757 US9608757W WO9639154A1 WO 1996039154 A1 WO1996039154 A1 WO 1996039154A1 US 9608757 W US9608757 W US 9608757W WO 9639154 A1 WO9639154 A1 WO 9639154A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotides
seq id
oligonucleotide
phosphorothioate
sequence
Prior art date
Application number
PCT/US1996/008757
Other languages
French (fr)
Inventor
Phillip Dan Cook
Glenn Hoke
Original Assignee
Isis Pharmaceuticals, 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
Priority to US08/466,692 priority Critical patent/US5654284A/en
Priority to US08/471,966 priority
Priority to US08/469,851 priority patent/US5587361A/en
Priority to US08/471,966 priority patent/US5661134A/en
Priority to US08/467,597 priority
Priority to US08/468,447 priority patent/US5576302A/en
Priority to US08/470,129 priority
Priority to US08/466,692 priority
Priority to US08/467,597 priority patent/US5607923A/en
Priority to US08/468,447 priority
Priority to US08/469,851 priority
Priority to US08/468,569 priority patent/US5620963A/en
Priority to US08/471,967 priority patent/US5599797A/en
Priority to US08/468,569 priority
Priority to US08/470,129 priority patent/US5635488A/en
Priority to US08/471,967 priority
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority claimed from KR1019970709017A external-priority patent/KR100257972B1/en
Publication of WO1996039154A1 publication Critical patent/WO1996039154A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1133Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against herpetoviridae, e.g. HSV
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/315Phosphorothioates

Abstract

Sequence-specific phosphorothioate oligonucleotides comprising nucleoside units which are joined together by either substantially all Sp or substantially all Rp phosphorothioate intersugar linkages are provided. Such sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are prepared by enzymatic or chemical synthesis.

Description

OLIGONUCLEOTIDES HAVING PHOSPHOROTHIOATE LINKAGES

OF HIGH CHIRAL PURITY

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Patent application Serial Number 08/297,703, filed August 29, 1994, which is a continuation of U.S. Patent application Serial Number 07/777,007, filed October 16, 1991, now abandoned. This application also is a continuation-in-part of U.S. Patent application Serial Number 08/058,023, filed May 5, 1993, which is a divisional application of U.S.

Patent application Serial Number 07/777,670, filed October 15, 1991 (issued as U.S. Patent 5,212,295, issue date May 18, 1993) , which is a continuation-in-part of U.S. Patent application Serial Number 07/777,007. Each of the above- mentioned applications is commonly assigned with this application, and the entire disclosures of each are herein incorporated by reference.

FIELD OF THE INVENTION

This invention is directed to sequence-specific phosphorothioate oligonucleotides comprising nucleosides joined by intersugar linkages, and to their synthesis and use. More particularly, the intersugar linkages linking the nucleosides of oligonucleotides of the present invention are substantially pure all Sp or all Rp chiral phosphorothioate linkages. Such oligonucleotides are prepared via chemical or enzymatic synthesis. They are especially well suited as diagnostics, therapeutics and research reagents. BACKGROUND OF THE INVENTION

Oligonucleotides are known to hybridize to single- stranded RNA or single-stranded DNA. Hybridization is the sequence-specific base pair hydrogen bonding of bases of the oligonucleotides to bases of target RNA or DNA. Such base pairs are said to be complementary to one another.

In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (TJ , a characteristic physical property of double helices, denotes the temperature (°C) at which 50% helical (hybridized) versus coil (unhy- bridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity) . Consequently, a reduction in UV absorption indicates a higher Tra. The higher the Tm, the greater the strength of the bonds between the strands.

Oligonucleotides can be used to effect enzymatic cleavage of a target RNA by using the intracellular enzyme RNase H. The mechanism of such RNase H cleavage requires that a 2' -deoxyribofuranosyl oligonucleotide hybridize to a target RNA. The resulting DNA-RNA duplex activates the RNase H enzyme and the activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the target RNA. Phosphorothioate oligo- nucleotides operate via this type of mechanism. However, for a DNA oligonucleotide to be useful for cellular activation of RNase H, the oligonucleotide must be reasonably stable to nucleases in order to survive in a cell for a time period sufficient for RNase H activation. For non-cellular uses, such as use of oligonucleotides as research reagents, such nuclease stability may not be necessary. - 3 -

Several publications of alder eϋ al . describe the interaction of RNase H and oligonucleotides. Of particular interest are: (1) Dagle et al . , Nucleic Acids Research 1990, 18, 4751; (2) Dagle et al . , Antisense Research And Development 1991, 1, 11; (3) Eder et al . , J. Biol . Chem. 1991, 266, 6472; and (4) Dagle et al . , Nucleic Acids Research 1991, 19, 1805. According to these publications, DNA oligonucleotides having both unmodified phosphodiester internucleoside linkages and modified phosphorothioate internucleoside linkages are substrates for cellular RNase H. Since they are substrates, they activate the cleavage of target RNA by RNase H. However, the authors further note that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also subject to exonuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonucleotide available for RNase H activation.

As described in references (1) , (2) and (4) , to stabilize oligonucleotides against nuclease degradation while still providing for RNase H activation, 2'-deoxy oligonucleotides having a short section of phosphodiester linked nucleotides positioned between sections of phosphoramidate, alkyl phosphonate or phosphotriester linkages were constructed. While the phosphoamidate- containing oligonucleotides were stabilized against exonuc- leases, in reference (4) the authors noted that each phos¬ phoramidate linkage resulted in a loss of 1.6°C in the measured Tm value of the phosphoramidate containing oligonu¬ cleotides. Such a decrease in the Tm value is indicative of a decrease in hybridization between the oligonucleotide and its target nucleic acid strand.

Applications of oligonucleotides as diagnostics, research reagents, and therapeutic agents require that the oligonucleotides be transported across cell membranes or taken up by cells, appropriately hybridize to target RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend partly on the initial stability of oligonucleotides towards nuclease degradation. Further, these functions depend on specificity of the oligonucleotide for a target RNA or DNA molecule.

A serious deficiency of oligonucleotides for these purposes is their susceptibility to enzymatic degradation by a variety of ubiquitous nucleases which may be intracellularly and extracellularly located. Unmodified, "wild type", oligonucleotides are not useful as therapeutic agents because they are rapidly degraded by nucleases. Therefore, modification of oligonucleotides for conferring nuclease resistance on them has been the primary focus of research directed towards the development of oligonucleotide therapeutics and diagnostics.

Modifications of oligonucleotides to enhance nuclease resistance has generally taken place on the sugar- phosphate backbone, particularly on the phosphorous atom. Phosphorothioates have been reported to exhibit resistance to nucleases. In addition, phosphorothioate oligonucleotides are generally more chemically stable than natural phosphodiester oligonucleotides. Phosphorothioate oligonucleotides also exhibit solubility in aqueous media. Further, phosphorothioate oligonucleotide-RNA heteroduplexes can serve as substrates for endogenous RNase H. Additionally, phosphorothioate oligonucleotides exhibit high thermodynamic stability. However, while the ability of an oligonucleotide to bind to a target RNA or DNA with fidelity is critical for its hybridization to the target RNA or DNA, modifications at the phosphorous atom of the oligonucleotides, while exhibiting various degrees of nuclease resistance, have generally suffered from inferior hybridization properties [Cohen, J.S., Ed., Oligonucleotides : Antisense Inhibi tors of Gene Expression (CRC Press, Inc., Boca Raton, FL, 1989].

One reason for this inferior hybridization may be the prochiral nature of the phosphorous atom. Modifications on the internal phosphorous atom of modified phosphorous oligonucleotides results in Rp and Sp stereoisomers. Modified phosphorus oligonucleotides obtained thus far, wherein the resulting molecule has nonsymmetrical substituents, have been racemic mixtures having 2n isomers, with n equal to the number of phosphorothioate intersugar linkages in the oligonucleotide. Thus, a 15-mer phosphorothioate oligonucleotide, containing 14 asymmetric centers has 214 or 16,384 diastereomers. In view of this, in a racemic mixture, only a small percentage of the oligonucleotides are likely to specifically hybridize to a target mRNA or DNA with sufficient affinity.

Chemically synthesized phosphorothioate oligo¬ nucleotides having chirally pure intersugar linkages had thus far been limited to molecules having only one or two diastereomeric intersugar linkages. Until recently, the effects of induced chirality in chemically synthesized racemic mixtures of sequence-specific phosphorothioate oligonucleotides had not been assessed since synthesis of oligonucleotides having chirally pure intersugar linkages had yet to be accomplished by automated synthesis. This was due to the non-stereospecific incorporation of sulfur during automated synthesis. For example, Stec et al . , J. Chromatography, 326:263 (1985) , synthesized certain oligonucleotide phosphorothioates having racemic intersugar linkages, however, they were able to resolve only the diastereomers of certain small oligomers having one or, at most, two diastereomeric phosphorous intersugar linkages.

However, Stec et al . [Nucleic Acids Res . , 19: 5883 (1991)] subsequently reported the automated stereocontrolled synthesis of oligonucleotides. The procedure described in the above-mentioned reference utilizes base-catalyzed nucleophilic substitution at a pentavalent phosphorothioyl center.

The synthesis of phosphorothioates having all Rp intersugar linkages using enzymatic methods has been investigated by several authors [Burgers and Eckstein, J". Biological Chemistry, 254:6889 (1979) ; Gupta et al . , J. Biol . Chem. , 256:7689 (1982) ; Brody and Frey, Biochemistry, 20:1245 (1981) ; and Eckstein and Jovin, Biochemistry, 2:4546 (1983)] . Brody et al . [Biochemistry, 21: 2570 (1982)] and Romaniuk and Eckstein, [J. Biol . Chem. , 257:7684 (1982)] enzymatically synthesized poly TpA and poly ApT phosphorothioates, while Burgers and Eckstein [Proc. Natl . Acad. Sci . U. S.A. , 75: 4798 (1978)] enzymatically synthesized poly UpA phosphorothioates. Cruse et al . [J. Mol . Biol . , 192:891 (1986)] linked three diastereomeric Rp GpC phosphorothioate dimers via natural phosphodiester bonds into a hexamer.

The relative ability of an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (TJ , a characteristic physical property of double helixes, denotes the temperature (°C) at which 50% helical versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity) . Consequently a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the binding of the strands. Non- Watson-Crick base pairing has a strong destabilizing effect on the Tm.

In a preliminary report [Stec, J.W., Oligonucleotides as Antisense Inhibi tors of Gene Expression : Therapeutic Implications, Meeting abstracts, June 18-21, 1989] , thymidine homopolymer octamers having all but one linkage being modified phosphate linkages ("all except one") Rp stereoconfiguration or "all except one" Sp stereoconfiguration in the intersugar linkages were formed from two thymidine methylphosphonate tetrameric diastereomers linked by a natural phosphodiester bond. It was noted that a Rp "all except one" methylphosphonate non- sequence-specific thymidine homooctamer, i.e. (dT)8 having all but one Rp intersugar linkage, formed a thermodynamically more stable hybrid (Tm 38°C) with a 15-mer deoxyadenosine homopolymer, i.e. (dA)15 than a hybrid formed by a similar thymidine homopolymer having "all except one" Sp configuration methylphosphonate linkages and of d(A) 15 (Tm <0°C) , i . e . a d(T)15 having all but one Sp intersugar linkage. A hybrid between (dT)8 having natural phosphodiester linkages, i . e . octathymidylic acid, and d(A)15 was reported to have a Tm of 14°C.

More recently, Ueda et al . [Nucleic Acids Research, 19:547 (1991)] enzymatically synthesized mRNAs intermittently incorporating Rp diastereomeric phosphoro¬ thioate linkages for use in translation systems. Ueda et al . employed T7 coliphane DNA having seventeen promoters and one termination site for T7 RNA polymerase. In vi tro synthesis by T7 RNA polymerase produced mRNAs having from several hundred to tens of thousands of nucleotides.

Backbone chirality may also affect the susceptibility of a phosphorothioate oligonucleotide-RNA heteroduplex to RNase H activity. The ability to serve as a template for RNAse H has significant therapeutic implications since it has been suggested that RNAse H causes cleavage of the RNA component in an RNA-DNA oligonucleotide heteroduplex. With oligonucleotides containing racemic mixtures of Rp and Sp intersugar linkages, it is not known if all phosphorothioate oligonucleotides can function equally as substrates for RNase H. For a variety of catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (an in-line mechanism) and inversion of configuration. Therefore, there may be only a small percentage of oligonucleotides in a racemic mixture that contain the correct chirality for maximum hybridization efficiency and termination of translation. Thus, increasing the percentage of phosphorothioate oligonucleotides that can serve as substrates for RNAse H in a heteroduplex will likely lead to a more efficacious compound for antisense therapy. To enhance hybridization fidelty, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired. Further, such phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages would lead to more efficacious therapeutic compounds. However, until now little success has been achieved in synthesizing such molecules. Therefore, simple methods of synthesizing phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired.

It has been recognized that nuclease resistance of oligonucleotides and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Oligonucleotides possessing nuclease resistance are also desired as research reagents and diagnostic agents.

SUMMARY OF THE INVENTION

In accordance with this invention, phosphorothioate oligonucleotides having all nucleoside units joined together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages are provided. Preferably, the phosphorothioate oligonucleotides of the present invention are complementary to at least a portion of the sequence of a target RNA or DNA.

Further, in accordance with the present invention, chemical and enzymatic methods of synthesizing sequence- specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are provided wherein said phosphorothioate oligonucleotides are comprised of at least 10 nucleoside units joined together by either substantially all Rp or substantially all Sp intersugar linkages. Preferably, the phosphorothioate oligonucleotides are comprised of about 10 to about 50 nucleoside units joined by substantially chirally pure intersugar linkages. More preferably, said phosphorothioate oligonucleotides are comprised of about 15 to about 30 nucleoside units joined by substantially chirally pure intersugar linkages. Most preferably, said phosphorothioate oligonucleotides are comprised of about 17 to 21 nucleoside units joined together by substantially chirally pure intersugar linkages. Said methods comprise combining sequence primers, templates, and an excess of all four chirally pure nucleoside 5'-0-(l- thiotriphosphates) . Said methods further include synthesizing complementary oligonucleotides by the addition of polymerase followed by cleavage of the primer from the complementary oligonucleotides. In addition, said methods are comprised of disassociating said complementary oligonucleotides from said template.

In alternative embodiments of the present invention methods of synthesizing sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages, sequence primers, templates and racemic mixtures of nucleoside 5'-0-(l- thiotriphosphates) are combined. Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages and which are complementary to the template are synthesized by the addition of polymerase and a selected metal ion. Oligonucleotides thus synthesized are dissociated from the template and primer. Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are useful for increasing the thermodynamic stability of heteroduplexes formed with target RNA and DNA and to elicit RNase H activity. Further, oligonucleotides of the present invention are useful for modulating the activity of RNA.

DETAILED DESCRIPTION OF THE INVENTION

The phosphorous atom in a phosphodiester linkage of an oligonucleotide can be described as being "pro¬ chiral." Once a non-bonding oxygen atom of the phosphodi- ester linkage is replaced or modified, a chiral sugar- phosphate linkage is generated. The resulting intersugar linkage is either an Sp intersugar linkage or an Rp intersugar linkage. Replacement of a non-bonding oxygen atom of the natural phosphodiester linkage with sulfur to obtain a phosphorothioate linkage results in the generation of a chiral center and affords Sp and Rp diastereomers.

Molecules wherein substantially all of the phosphorous atoms in the sugar backbone are either Sp or Rp are referred to herein as chirally pure.

Ribonucleoside- (NTPαS) and 2' -deoxyribonucleo- side-5' -0- (1-thiotriphosphates) (dNTPαS) have been synthesized as Sp and Rp racemic mixtures using the methodology of Ludwig and Eckstein [J. Org. Chem. , 631 (1989)] . In this exemplary synthetic scheme, unprotected nucleosides can be reacted with 2-chloro-4H-l,3,2- benzodioxaphosphorin-4-one, which phosphitylates the 5'- hydroxyl group. Subsequent reaction with pyrophosphate yields cyclic triphosphate derivatives which are reactive to sulfur, yielding mixtures of Rp and Sp nucleoside 5'-0-(l- thiotriphosphates) , i.e. α-thiotriphosphates. The products can be purified by DEAE-Sephadex chromatography and identi¬ fied by NMR spectroscopy (by characteristic Rp or Sp chemical shifts) .

As is shown in the examples below, pure Rp and Sp nucleoside-5' -0- (1-thiotriphosphates) diastereomers can be readily isolated on a preparative scale using, for example, reverse phase HPLC chromatography. Such HPLC-isolated nucleotide diastereomers can be further characterized by analytical HPLC comparisons with commercial samples of such Rp and Sp nucleoside 5' -0- (1-thiotriphosphates) diastereome- rs.

Enzymatic synthesis of sequence-specific natural oligonucleotides, i.e. natural phosphodiester oligonucleotides, can be effected by the use of an appropriate nuclease in the presence of a template and primer. In a like manner, racemic mixtures of phosphorothioate oligonucleotides having chirally mixed intersugar linkages can be synthesized. According to the present invention, such enzymatic synthesis can also be expanded to include the synthesis of sequence specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages by utilizing enantiomerically pure all-Sp or all-Rp nucleoside 5'-0-(l- thiotriphosphates) as substrates for appropriate nucleases in the presence of a sequence-specific template and a primer. For example, commercially available DNA polymerase Sequenase™ (U.S. Biochemical, Inc., Cleveland, OH) may be used to synthesize phosphorothioate oligonucleotides using a phosphodiester oligonucleotide template and a racemic phosphorothioate oligonucleotide primer. Using this polymerase both phosphodiester and phosphorothioate primers may be extended. Yields of enzymatically synthesized phosphoro¬ thioate oligonucleotides can be optimized by repetitive additions of template and primer, by repetitive additions of polymerase, by repetitive additions of nucleoside triphosphates or by combinations of some or all of these. For instance, repetitive additions of template and primer results in maximizing yields via an enzymatic cascade. Further optimization can be achieved by pre-hybridization of template and primer together in system buffer, followed by cooling and addition of nucleoside triphosphates and polymerase.

A suitable polymerase may be selected to yield either DNA or RNA phosphorothioate oligonucleotides. Such polymerases include but are not necessarily limited to T7 DNA polymerase, modified T7 DNA polymerases such as the above referenced Sequenase™, E. coli DNA polymerase, DNA poly Klenow fragment polymerase, M. luteus polymerase, T4 bacteriophage polymerase, modified T4 DNA polymerase, T7 RNA polymerase and E. coli RNA polymerase.

The enzymatic synthesis proceeds with inversion of configuration about the chiral center of the phosphorous atom. Thus, use of all Sp α-thiotriphosphates yields substantially all Rp phosphorothioate oligonucleotides while use of all Rp α-thiotriphosphates yields substantially all Sp phosphorothioate oligonucleotides. In an alternate embodiment of the invention, phosphorothioate oligonucleotides may be synthesized from racemic mixtures of nucleoside-5' -0- (1-thiotriphosphates) utilizing metal ions in reaction solutions to promote preferential incorporation of one or the other of the chiral α-thiotriphosphates. As noted above, polymerase synthesis of phosphorothioate oligonucleotides is accomplished with inversion of configuration about the chiral center of the precursor nucleoside-α-thiotriphosphate. While not wishing to be bound by theory, it is believed that optimization of an all Rp configuration may be accomplished by addition of a high concentration of magnesium ion in the reaction buffer utilizing, for instance, an E. coli polymerase. In a like manner, again while not wishing to be bound by theory, an all Sp configuration might be obtained by utilizing a high manganese ion concentration in the reaction buffer. In accordance with the present invention, "substantially all" is meant to include all oligonucleotides in which at least 75% of the intersugar linkages are chirally pure. More preferably, oligonucleotides having from about 85% to about 100% chirally pure intersugar linkages are substantially chirally pure. Most preferably, oligonucleotides having from about 95% to about 100% chirally pure intersugar linkages are substantially chirally pure.

In the context of this invention, the term "phosphorothioate oligonucleotide" includes phosphorothioate oligonucleotides formed from naturally occuring bases, sugars and phosphorothioate linkages. Naturally occuring bases include adenine, guanine, cytosine, thymine and uracil. Natural sugars include β-D-ribofuranosyl and β-D- 2' -deoxy-eryt ro-pentofuranosyl. To the extent that nucleoside-5' -0- (l-thiotriphosphate) analogs are substrates for suitable polymerases, "phosphorothioate oligonucleotides" also include modified bases or modified sugars incorporated within the phosphorothioate nucleotide units of the oligonucleotides. Modified bases of the oligonucleotides of this invention include adenine, guanine, adenine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8- substituted guanines, other aza and deaza uracils, other aza and deaza thymidines, other aza and deaza cytosines, other aza and deaza adenines, other aza and deaza guanines, 5- trifluoromethyl uracil and 5-trifluoro cytosine. The sugar moiety may be deoxyribose or ribose. The oligonucleotides of the invention may also comprise modified nucleobases or nucleobases having other modifications consistent with the spirit of this invention, and in particular modifications that increase their nuclease resistance in order to facilitate their use as therapeutic, diagnostic or research reagents. Oligonucleotides of the invention can be utilized as diagnostics, therapeutics and as research reagents. They can be utilized in pharmaceutical compositions by adding an effective amount of an oligonucleotide of the invention to a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism can be contacted with an oligonucleo- tide of the invention having a sequence that is capable of specifically hybridizing with a strand of target nucleic acid that codes for the undesirable protein.

The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. In general, for therapeutics, a patient in need of such therapy is administered an oligonucleotide in accordance with the invention, commonly in a pharmaceutically acceptable carrier, in doses ranging from 0.01 μg to 100 g per kg of body weight depending on the age of the patient and the severity of the disease state being treated. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient, and may extend from once daily to once every several years. Following treatment, the patient is monitored for changes in his/her condition and for alleviation of the symptoms of the disease state. The dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, or if the disease state has been ablated.

In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on ECsos found to be effective in in vi tro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every several years.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every several years.

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal) , oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Phosphorothioate oligonucleotides of the present invention can be contrasted with both natural phosphodiester oligonucleotides and racemic phosphorothioate nucleotides as to their effects on hybridization, nuclease resistance and RNAse H activity. In like manner, pure phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages may also be assessed for their ability to increase effectiveness of therapy in in vivo test systems. Such increase in effectiveness of therapy might include attributes such as pharmacokinetics or metabolism, toxicology, disposition (i.e. absorption and distribution), and species comparisons.

Homopolymers having all Rp or all Sp intersugar linkages have been useful for initial studies of stability and other characteristics. However, these oligonucleotides have little use therapeutically as they are not specific for target molecules. Phosphorothioate oligonucleotides having specific sequences are necessary in order to specifically hybridize to target nucleic acids.

Sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are useful to increase the thermodynamic stability of heteroduplexes with target RNA and DNA and to elicit RNase H activity.

Radiolabeling can be used to assist in the identification of phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages. For racemic phosphorothioate oligonucleotides synthesized on an automated synthesizer, [35S] (radiolabeled elemental sulfur) can be used for oxidation of the hydrogen-phosphonate oligomers obtained from the synthesizer. Labeling of enzymatically synthesized phosphorothioate oligonucleotides can be accomplished with [α-32P]ATP and ligase or [α-35S]ATPs in the polymerase reaction. Also, radiolabeled nucleoside triphosphates can be used in probe and sequencing analysis. Autoradiograms are prepared in standard manners. Templates of the present invention are most preferably areas of nucleic acid sequence which direct synthesis of disease-potentiating proteins. Short oligonucleotides that base pair to a region of said template oligonucleotide act as primers which form the starting point for oligonucleotide synthesis by polymerases.

Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages may be synthesized using a primer which may be selected to have a site thereon that is susceptible to nuclease cleavage, for example, restriction endonuclease cleavage. Said cleavage site may be located at the 3' end of said primer. Cleavage at said site by an appropriate restriction endonuclease results in oligonucleotides deriving a first 5' end nucleoside from said primer. Additional nucleosides of said phosphorothioate oligonucleotides of the present invention are those nucleoside chiral thiotriphosphates added via enzymatic means.

By selecting appropriate restriction nucleases in conjunction with selected primers, various 5' -terminal nucleosides of desired phosphorothioate oligonucleotides are appropriately positioned at the 5' end of a phosphorothioate nucleotide. Thus, any endonuclease recognition site can be designed as long as the staggered cut results in one nucleoside from the primer being the first 5' nucleoside of the newly synthesized sequence specific phosphorothioate oligonucleotide of the invention. This results in the generation of different nucleosides on 5' ends of enzymati¬ cally synthesized phosphorothioate oligonucleotides of the invention.

Upon completion of enzymatic extension of said primer on an appropriate template of a desired sequence, phosphorothioate oligonucleotides of the invention may be released from said primer by use of appropriate nuclease. For example, for incorporation of a guanosine nucleoside at the 5' end of desired phosphorothioate oligonucleotides, a primer having an CTGCAG sequence at its 3' terminal end may be used. Use of a Pst 1 restriction nuclease then may cleave the A-G linkage. The guanosine nucleoside component of this A-G linkage may thus incorporated as a 5' terminal nucleoside of desired phosphorothioate oligonucleotides. Other restriction endonuclease include but are not limited to BamHl, Smal and HinD III restriction endonucleases. Oligonucleotides still associated with said template may be dissociated from said template and then purified by gel electrophoresis and/or chromatography. For example, suitable purification can be accomplished utilizing standard polyacrylamide/urea gel electrophoresis coupled with SepPac (Millipore, Miford, MA) chromatography. Another useful chromatographic technique that may be employed is HPLC chromatography.

Chiral phosphorothioate oligonucleotides of the present invention may also be chemically synthesized via 1,3,2-oxathiaphospholane intermediates as described by Stec et al . [Nucleic Acids Res . , 19:5883 (1991)] and Stec and

Lesnikowski [Methods in Molecular Biology, S. Agrawal, Ed., Volume 20, p. 285, 1993] .

Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages which are synthesized according to methods of the present invention may be analyzed by a number of methods. For example, configuration analysis of resulting sequence-specific phosphorothioate oligonucleotides having subtantially chirally pure all Sp or all Rp intersugar linkages may be determined by the use of [31P] NMR chemical shifts. Such chemical shifts have been used to identify the Rp epimer of a phosphorothioate dinucleotide [Ludwig and Eckstein, J. Org. Chem. , 631-635 (1989)] .

The fidelity of sequences of phosphorothioate oligonucleotides of the invention can be determined using the sensitivities of heteroduplexes to SI nuclease.

The sequence of the phosphorothioate oligonucleotides can be further substantiated by labeling the 3'hydroxyl groups of phosphorothioate oligonucleotides with [alpha-32P] cordycepin triphosphate, i.e. 3' -deoxyadeno- sine-5' -triphosphate. The resultant oligonucleotides may be subjected to enzymatic degradation.

The relative ability of phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages to bind to complementary strands is compared by determining the melting temperature of a hybridization complex of a phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages and its complementary strand. The melting temperature (T , a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coiled (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity) . Consequently a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the binding of the strands. Non Watson-Crick base pairing has a strong destabilizing effect on the Tm. Consequently, as close to optimal fidelity of base pairing as possible is desired to have optimal binding of an oligonucleotide to its target RNA.

Phosphorothioate oligonucleotides of the invention can also be evaluated for their resistance to the degradative ability of a variety of exonucleases and endonucleases. Phosphorothioate oligonucleotides may be treated with nucleases and then analyzed, as for instance, by polyacrylamide gel electrophoresis (PAGE) followed by staining with a suitable stain such as Stains All™ (Sigma Chem. Co., St. Louis, MO) . Degradation products may be quantitated using laser densitometry.

Fetal calf and human serum may be used to evaluate nucleolytic activity on phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages. For instance, a phosphorothioate oligonucleotide having substantially all Rp intersugar linkages may be evaluated in this manner. Testing on combinations of 3' or 5' end capped (having one or several phosphorothioate linkages per cap) molecules may be used to establish a combination that yields greatest nuclease stability. Capping can be effected by chemically synthesizing the cap portion of a sequence using purified Rp monomers followed by incorporation of said cap into oligonucleotides on the DNA synthesizer. Analysis involving capping can determine the importance of chirality on nucleolytic stability and the number of linkages required to obtain maximum stability.

The sensitivity of phosphorothioate oligonucleotide-RNA heteroduplexes to the catalytic activity of RNase H can also be assessed. A phosphorothioate oligo¬ nucleotide can be incubated with a radiolabeled target mRNA (synthesized as for instance via T7 RNA polymerase) at various temperatures for hybridization. Heteroduplexes can then be incubated at 37°C with RNase H from E. coli according to the procedure of Minshull and Hunt [Nuc. Acid Res . , 6433 (1986)] . Products may then be assessed for RNase H activity by Northern Blot analysis wherein products are electrophoresed on a 1.2% agarose/formaldehyde gel and transferred to nitrocellulose. Filters may then be probed using a random primer [32P] -labeled cDNA complementary to target mRNA and quantitated by autoradiography. Comparisons between different phosphorothioate analogs can be made to determine the impact of chirality on the ability to act as a substrate for RNase H when complexed to RNA.

Comparisons of the susceptibility of heteroduplexes to the catalytic action of E. coli RNase H and mammalian RNAse H can be performed. Heteroduplexes can be incubated in rabbit reticulocyte lysates under conditions of translation and assayed via Northern blot analysis for catalytic cleavage of mRNA by endogenous RNase H. This allows for determination of the effects of chirality on mammalian RNAse H activity.

Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can also be evaluated for inhibition of gene expression in cell culture model systems. To determine if a phosphorothioate oligonucleotide having substantially pure chirally pure intersugar linkages is more potent or a more specific inhibitor of gene expression, a phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages designed to target reporter genes may be synthesized and tested in cell culture models of gene expression. The use of the vector pSV2CAT has previously been described to measure antisense effects on gene expression [Henthorn et al . , Proc . Na tl .Acad . Sci . U. S . A . , 85:6342 (1988)] . This vector contains the bacterial chloramphenicol acetyl transferase gene under regulatory controls of the SV40 promoter. Utilizing a 15-mer phosphorothioate oligonucleotide having all Rp intersugar linkages of a sequence complementary to the initiation of translation of the CAT mRNA, pSV2CAT may be transfected into HeLa cells and, following treatment of the cells for 48 hr with a phosphorothioate oligonucleotide having all Rp intersugar linkages, CAT activity may then be assayed in the cells. The activity of a phosphorothioate having substantially chirally pure intersugar linkages in inhibition of gene expression may then be compared directly with a chemically synthesized random phosphorothioate having diastereomeric intersugar linkages and natural phosphodiester oligonucleotides of the same sequence. The vector pSV2APAP [Marcus-Sekura et al . , Nucleic

Acids Research, 15:5749 (1987)] contains the mammalian placental alkaline phosphatase gene (PAP) . This can also be used as a reporter for measuring antisense effects on gene expression. PAP has advantages over CAT as a reporter gene in that it is a mammalian gene, rather than a bacterial gene that contains introns and other RNA processing signals. It is presently believed that PAP expression mimics more closely the events in natural mammalian gene expression. A 15-mer phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages as described above for the CAT mRNA can be examined in parallel with chemically synthe¬ sized racemic phosphorothioate and natural phosphodiester oligonucleotides having similar sequences. The PAP and CAT reporter constructs are used as controls in reciprocal experiments to test for non-specific effects on gene expression. Additionally, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can be evaluated as to their ability to act as inhibitors of RNA translation in vivo . Various therapeutic areas can be targeted for such manipulation by oligonuclotides of the present invention. One therapeutic area is hepatitis caused by Hepatitis C virus (HCV) . The following phosphorothioate oligonucleotides have application in the treatment of HCV hepatitis: Oligo # 259, CCTTTCGCGACCCAACACTA (SEQ ID N0:1), Oligo # 260, GCCTTTCGCGACCCAACACT (SEQ ID N0:2) , Oligo # 270, GTACCACAAGGCCTTTCGCG (SEQ ID NO:3), Oligo # 330, GTGCTCATGGTGCACGGTCT (SEQ ID NO:4) and Oligo # 340, TTTAGGATTCGTGCTCATGG (SEQ ID NO:5) . Another therapeutic area inflammatory diseases mediated by intercellular cell adhesion molecule (ICAM-1) . Oligonucleotides having application in the treatment of inflammatory diseases include: ISIS-2302, GCCCAAGCTGGCATCCGTCA (SEQ ID NO:6) . Another therapeutic area includes infections caused by cytomegalovirus (CMV) . ISIS-2922 is a phosphorothioate oligonucleotide having application in the treatment of CMV retinitis, and has the sequence GCGTTTGCTCTTCTTCTTGCG (SEQ ID NO:7) . Another therapeutic area includes cancers mediated by protein kinase C-α (PKC-α) . ISIS-3521 is a phosphorothioate oligonucleotide having application in the treatment of such cancers, and has the sequence

GTTCTCGCTGGTGAGTTTCA (SEQ ID NO:8) . A further therapeutic area includes C-raf kinase-mediated cancers. ISIS-5132 is a phosphorothioate oligonucleotide having application in the treatment of such cancers, and has the sequence TCCCGCCTGTGACATGCATT (SEQ ID NO:9) . A still further therapeutic area includes cancers mediated by Ha-ras or Ki- ras . The following phosphorothioate oligonucleotides have application in the treatment of such cancers: ISIS-2503, TCCGTCATCGCTCCTCAGGG (SEQ ID NO.IO), ISIS-2570, CCACACCGACGGCGCCC (SEQ ID N0:11), and ISIS-6957,

CAGTGCCTGCGCCGCGCTCG (SEQ ID NO:12) . Another therapeutic area includes AIDS and other related infections mediated by HIV. ISIS-5320 is a phosphorothioate oligonucleotide having application in the treatment of AIDS, and has the sequence TTGGGGTT (SEQ ID NO:17) . In the above sequences, individual nucleotide units of the oligonucleotides are listed in a 5' to 3' direction from left to right.

In the context of this invention, "hybridization" shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotide units. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein, also refers to sequence complementarity between two nucleotide units. For example, if a nucleotide unit at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide unit at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotide units which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide and the target RNA or DNA. It is understood that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target RNA or DNA molecule interferes with the normal function of the target RNA or DNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vi tro assays, under conditions in which the assays are performed. In particular, oligonucleotides of the invention may be used in therapeutics, as diagnostics, and for research as is specified in the following United States patent applications assigned to the assignee of this invention These applications are entitled: Compositions and Methods for Modulating RNA Activity, Serial No. 463,358, filed 1/11/90; Antisense Oligonucleotide Inhibitors of Papilloma Virus, Serial No. 445,196 Filed 12/4/89; Oligonucleotide Therapies for Modulating the Effects of Herpesvirus, Serial No. 485,297, Filed 2/26/90; Reagents and Methods for Modulating Gene Expression Through RNA Mimicry Serial No. 497,090, Filed 3/21/90; Oligonucleotide Modulation of Lipid Metabolism, Serial No. 516,969, Filed 4/30/90; Oligonucleotides for Modulating the Effects of Cytomegalovirus Infections, Serial No. 568,366, Filed

8/16/90; Antisense Inhibitors of the Human Immunodeficiency Virus, Serial No. 521,907, Filed 5/11/90; Nuclease Resistant Pyrimidine Modified Oligonucleotides for Modulation of Gene Expression, Serial No.558,806, Filed 7/27/90; Novel Polyamine Conjugated Oligonucleotides, Serial No. 558,663, Filed 7/27/90; Modulation of Gene Expression Through Interference with RNA Secondary Structure, Serial No. 518,929, Filed 5/4/90; Oligonucleotide Modulation of Cell Adhesion, Serial No. 567,286, Filed 8/14/90; Inhibition of Influenza Viruses, Serial No. 567,287, Filed 8/14/90;

Inhibition of Candida, Serial No. 568,672, Filed 8/16/90; and Antisense Oligonucleotide Inhibitors of Papillomavirus, Serial No. PCT/US90/07067, Filed 12/3/90. These applications disclose a number of means whereby improved modulation of RNA and DNA activity may be accomplished through oligonucleotide interaction. In that the specific sequences disclosed therein may be used in conjunction with the present invention, the disclosures of the foregoing United States patent applications are incorporated herein by reference.

The following examples are illustrative and are not meant to be limiting of the present invention. EXAMPLE 1

ISOLATION OF ALL Sp OR ALL Rp 5' -O- (1-THIOTRIPHOSPHATE)

NUCLEOSIDE

5' -0- (l-thiotriphosphate) deoxynucleosides and ribonucleosides are isolated using C-18 reverse phase high performance liquid chromatography (HPLC) using columns packed with ODS Hypersil (Shandon Southern, Runcon, UK) and eluted with an isocratic mixture of solvent A (30 mM potassium phosphate containing 5 mM tetrabutylammonium ion, pH 7.0) and solvent B (5 mM tetrabutylammonuium hydroxide in methanol) . Alternatively, effective separation is achieved using 100 mM triethylammonium bicarbonate, pH 7.5, containing a linear gradient of acetonitrile from 0% to 15% over 20 minutes. To establish the purity of such HPLC separated enantiomers the HPLC separated Sp and Rp deoxynucleotide enantiomers are compared to commercially available deoxy¬ nucleoside 5' -O- (1-thiotriphosphates) available from E.I. Dupont, Wilmington, DE.

EXAMPLE 2

SYNTHESIS OF PHOSPHOROTHIOATE EXTENSION HAVING SUBSTANTIALLY

ALL Rp INTERSUGAR LINKAGES OF A RACEMIC PHOSPHOROTHIOATE

OLIGONUCLEOTIDE Enzymatic synthesis of an all Rp phosphorothioate extension of a racemic phosphorothioate oligonucleotide primer is effected using the modified T7 DNA polymerase I, Sequenase™ (U.S. Biochemicals Corp, Cleveland, OH) . This T7 DNA polymerase is used to extend an 18 mer phosphorothioate oligonucleotide primer hybridized to a 21-mer natural phosphodiester oligonucleotide. 30 picomoles (pmol) of primer and template in a IX Sequenase™ reaction buffer (U.S. Biochemicals Corp., Cleveland, OH) (final vol 10 μL) are heated for 5 minutes at 95°C and slowly cooled to room temperature. 180 pmol of deoxy 5' - [α-35S] cytidine triphosphate and Sequenase™ enzyme (U.S. Biochemicals Corp., Cleveland, OH) are added and incubated at 37°C for 20 minutes. The product is analyzed via polyacrylamide gel electrophoresis (PAGE) using a 20% polyacrylamide/7 M urea denaturing gel. The autoradiograph of the product is compared to a control reaction absent primer/template. The final product is subjected to further characterization by, for example, enzymatic degradation. One such degradation is snake venom phosphatase degradation. A snake venom phospha- tase degradation of dinucleoside monophosphorothioate synthesized using E. coli DNA polymerase I shows the dinucleoside to be of the Rp configuration.

EXAMPLE 3

SYNTHESIS OF PHOSPHOROTHIOATE CGACTATGCAAGTAC (SEQ ID NO:13) OLIGONUCLEOTIDE HAVING SUBSTANTIALLY PURE Rp INTERSUGAR LINKAGES

A large scale enzymatic synthesis of sequence specific all Rp phosphorothioate oligonucleotide was effected utilizing a 55-mer natural phosphodiester template and a 41-mer natural phosphodiester primer. The template sequence was:

GTACTTGCATAGTCGATCGGAAAATAGGGTTCTCATCTCCCGGGATTTGGTTGAG (SEQ ID NO:14) . The primer sequence was: CTCAACCAAATCCCGGGAGATGAGAACCCTATTTTCCGATC (SEQ ID NO:15) . The template was selected to have a sequence complementary to a desired specific CGACTATGCAAGTAC (SEQ ID NO:13) sequence. A Sequenase™ buffer (U.S. Biochemicals Corp., Cleveland, OH) diluted from 5X to IX was used. The template and primer, both at concentrations of 20 nM are added to 40 μL of this buffer. The template and primer were hybridized at 95°C for 5 minutes and cooled to room temperature. After cooling the buffer was adjusted to 7 mM DTT. 20 μL of 1:8 diluted Sequenase™ enzyme and 320 μM each of Sp GTPαS, CTPαS, ATPαS and TTPαS were then added. The reaction solution was adjusted to 140 μL with H20. It was incubated at 37°C for 18 hours. The reaction solution was extracted 2X with a like volume of phenol in a standard manner and precipated in a standard manner by treatment with 2.5 volumes of 100% ethanol at -20°C, peltized, washed with 500 μL of 70% ethanol, peltized again and dried. The precipitate was suspended in 20 μL H20 for 30 minutes then adjusted to 1 mM CaCl2, 25 mM Tris HCl pH 8.0 in 40 μL H20. The solution was maintained at 95°C for 5 minutes and snap cooled, i.e. very quickly cooled with ice. The template and primer were removed from the synthesized oligonucleotide by the addition of 4.6 μM DNase I and incubation at 37°C for 10 minutes. The reaction mixture was phenol extracted 2X and precipitated with ethanol as above. The precipate was resuspended in H20 and purfied using 20% polyacrylamide/7 M urea gel electrophoresis coupled with SepPak™ chromatography (Millipore, Milford, MA) .

In an alternate synthesis, Pst 1 restriction nuclease (Life Technologies, Inc., Gaithersburg, MD) was used to cleave the primer-bound phosphorothioate oligonu- cleotide at the restriction site. The desired

CGACTATGCAAGTAC (SEQ ID NO:13) phosphorothioate oligonucleo¬ tide was purified using polyacrylamide/7 M urea gel electrophoresis coupled with SepPak™ chromatography (Milli¬ pore, Milford, MA) . Yields were optimized using enzymatic cascade effected by repetitive template-primer addition throughout the reaction. The cascade augmented synthesis yielded 75 A260 units of the CGACTATGCAAGTAC (SEQ ID NO:13) all Rp configuration phosphorothioate oligonucleotide from a 20 mL reaction.

EXAMPLE 4

SYNTHESIS OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING A RACEMIC MIXTURE OF INTERSUGAR LINKAGES USING AUTOMATED DNA SYNTHESIS.

Oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using hydrogenphosphonate chemistry in a standard manner [Agrawal et al., Proc . Natl . Acad. Sci . U. S. A . , 85:7079 (1988)] . After the final coupling step, the phosphorothioate linkages are generated by oxidizing the bound oligomer with sulfur in carbon disulfide/triethylamine/ pyridine. After sulfur oxidation, standard deblocking procedures with ammonium hydroxide are used to release the oligonucleotides from the support and remove base blocking groups. The phosphorothioate oligonucleotides are purified by oligonucleotide purification column (OPC; ABI, Foster City, CA) chromatography and HPLC, using a Beckman System Gold HPLC. The HPLC-purified oligonucleotides are then precipitated with ethanol and assessed for final purity by gel electrophoresis on 20% acrylamide/7 M urea or by analytical HPLC. The authenticity of the oligonucleotide sequence was assessed by oxidation with iodine in pyridine/water and standard sequencing methods. These oligonucleotides contain a mixture of all possible combinations of Rp and Sp isomers at each phosphorous linkage.

EXAMPLE 5

SYNTHESIS OF COMPLEMENTARY DNA OR RNA SEQUENCES USING T7 RNA POLYMERASE FOR THERMODYNAMIC AND KINETIC HYBRIDIZATION ANALYSIS.

The synthesis of short complementary DNA oligonucleotides of natural phosphodiester linkages was performed utilizing standard automated synthesis on an ABI model 380B DNA Synthesizer. The oligonucleotides of correct length were purified by HPLC and sequenced by standard techniques. T7 RNA polymerase was use for the synthesis of short, complementary RNA oligonucleotides for hybridization analysis. A large amount of T7 RNA polymerase at high concentrations was needed for the many cycles of initiation required to synthesize short RNAs. Due to this requirement, the T7 RNA polymerase was derived from a strain of E. coli that contained a T7 RNA polymerase expression vector, BL21/pAR1219, obtained from Brookhaven National Laboratory (Upton, NY) . The isolation yielded approximately 300,000 to 500,000 units of T7 RNA polymerase from 2 L of cells, absorbance value = 1.2 A600. This was sufficiently concentrated for synthesis of short (10-30 nucleotides) RNA species. For synthesis, a T7 promoter and a template con¬ taining the complementary target sequence and T7 promoter hybridization sequence were synthesized using the ABI synthesizer (ABI, Foster City, CA) . Template and promoter were purified by HPLC to ensure that the correct species was present for enzymatic synthesis. Synthesized products were purified on a 20% polyacrylamide/8 M urea gel and sequenced by standard procedures.

EXAMPLE 6 THERMAL DENATURATION

Oligonucleotides (either phosphorothioate oligonucleotides of the invention or otherwise) were incubated with either the complementary DNA or RNA oligonucleotides at a standard concentration of 4 μM for each oligonucleotide in 100 mM ionic strength buffer (89.8 mM NaCI, 10 mM Na-phosphate, pH 7.0, 0.2 mM EDTA) . Samples were heated to 90°C and the initial absorbance taken using a Guilford Response II spectrophotometer (Corning) . Samples were then slowly cooled to 15°C and the change in absorbance at 260 nm monitored during the heat denaturation procedure. The temperature was elevated 1 degree/absorbance reading and the denaturation profile analyzed by taking the first derivative of the melting curve. Data was also analyzed using a two-state linear regression analysis to determine the Tm and delta G. The results of these tests are shown in Table 1. TABLE 1 THERMAL DENATURATION

Sequence SEQ ID NO: Complement T_

Natural phosphodiester CGA CTA TGC AAG TAC 13 DNA 53.2

CGA CTA TGC AAG TAC 13 RNA 46.2

Phosphorothioate with racemic intersugar linkages

CGA CTA TGC AAG TAC 13 DNA 46.0 CGA CTA TGC AAG TAC 13 RNA 36.5

Phosphorothioate with chirally pure intersugar linkages

CGA CTA TGC AAG TAC 13 DNA 45.5

CGA CTA TGC AAG TAC 13 RNA 41.5 GA CTA TGC AAG TAC 16 DNA 44.5

GA CTA TGC AAG TAC1 16 RNA 40.0

EXAMPLE 7

SYNTHESIS OF RADIOLABELED OLIGONUCLEOTIDES

Filter binding assays are utilized to quantitate the binding stringencies of various phosphorothioate oligonucleotides, i . e . their tendencies to hybridize and form heteroduplexes with DNA or RNA. These assays require radiolabeled oligonucleotides.

Phosphorothioate oligonucleotides having all Rp intersugar linkages are synthesized by enzymatic methods from [35S] -monomers that have been purified from Sp monomers. For automated synthesis of phosphorothioate oligonucleotides containing mixed chirality intersugar linkages, oligonucleotides are synthesized containing hydrogen phosphonates and then sulfurized in the presence of elemental [35S] in a pyridine/carbon disulfide mixture. The resulting radiolabeled phosphorothioate oligonucleotide can be purified by OPC chromatography and HPLC. Target mRNA are applied to nitrocellulose filters and baked at 80°C for 2 hours, blocked and then hybridized with the radiolabeled phosphorothioate oligonucleotide. Binding stringency is assessed by quantitating radiolabeled oligonucleotide eluted from the filters after increases in temperature or increases in the ionic strength of an eluting buffer, as for instance, Tris NaCI buffer. Eluted oligonucleotides are also assessed for their mobility in an anion exchange HPLC protocol isocratically utilizing phosphate buffer. Results are compared to the mobility of standard oligonucleotides prepared having racemic mixtures of intersugar linkages.

EXAMPLE 8 NUCLEASE DIGESTION

Determination of the rate of nuclease degradation of the phosphorothioate oligonucleotides in media containing 10% fetal calf serum (FCS) was carried out in Dulbecco's Modified Essential Medium (DMEM) containing 10% heat inactivated FCS. Heat inactivation of the FCS was carried out at 55°C for 1 hour prior to addition to media. Oligonucleotides having racemic and chirally pure intersugar linkages were separately tested for resistance to nuclease digestion. 66 μg/mL of each oligonucleotide were separately added to medium and incubated at 37°C, at the time intervals indicated in Table 2. 15 μL Aliquots were removed and added to 15 μL of 9 M urea in 0.1 M Tris-HCl (pH 8.3), 0.1 M boric acid and 2 mM EDTA. Aliquots were mixed by vortex and stored at -20°C. Polyacrylamide gel electrophoresis (PAGE) analysis was on 20% polyacrylamide/7 M urea slab gels. Following electrophoresis, gels were stained using "Stains All" (Sigma Chem. Co., St. Louis, MO) . Following de- staining, gels were analyzed via laser densitometry using an UltraScan XL device (Pharmacia LKB Biotechnology, Uppsala, Sweden) . Integrations were performed and the data presented as the percentage decrease from full length (n) prior to incubation to n-1. These results are shown in Table 2 for the oligonucleotide sequence CGACTATGCAAGTAC (SEQ ID NO:6) having Rp chirally pure intersugar linkages.

TABLE 2 NUCLEASE DIGESTION Incubation in 10% fetal calf serum

Digestion of oligonucleotide of length n to length n-1

Time (hours) Phosphorothioate with Phosphorothioat with with racemic intersuσar chirally pure linkages intersugar linkages 0 0 0

1 44 10

2 45 10 4 54 12

24 70 44 48 70 62

As is evident from Table 2, the phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages showed greater resistance to nuclease degradation than did the phosphorothioate oligonucleotide having racemic intersugar linkages.

EXAMPLE 9

RNASE H ANALYSIS

Phosphorothioate oligonucleotides having racemic and substantially chirally pure intersugar linkages were analyzed for susceptibility to RNase H. Oligonucleotides (2-fold molar excess to RNA) and 5 μg (3.1 kb) in vi tro synthesized mRNA (using T7 RNA polymerase promoter) were incubated in 5 μL RNase H hybridization buffer for 30 minutes at 60°C. Samples were slowly cooled to room temperature and then adjusted to 3.7 mg/mL BSA, 20 units E. coli RNase H (Promega) , 142 mM DTT, 150 mM KCl, and 3 mM MgCl2. Samples were incubated for 30 minutes at 37°C. Samples were then extracted with phenol, precipitated with ethanol, and analyzed by electrophoresis on 1.2% agarose gels following ethidium bromide staining. Markers were run on gels concurrently with the samples to determine approximate length of RNA samples. EXAMPLE 10

A patient suffering from hepatitis caused by HCV is treated with Oligo # 259 (SEQ ID NO:l) , Oligo # 260 (SEQ ID NO:2) , Oligo # 270 (SEQ ID NO:3) , Oligo # 330 (SEQ ID NO:4) or Oligo # 340 (SEQ ID NO:5) , each of which are synthesized according to the procedure of Example 3 or Example 19. 1-1000 μg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

EXAMPLE 11

A patient suffering from an inflammatory disease mediated by ICAM-1 is treated with ISIS-2302, an oligonucleotide synthesized according to Example 3 or

Example 19, and having the sequence GCCCAAGCTGGCATCCGTCA (SEQ ID NO:6) . 1-1000 μg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

EXAMPLE 12

A patient suffering from retinitis caused by cytomegalovirus is treated with ISIS-2922, an oligonucleotide synthesized according to Example 3 or

Example 19, and having the sequence GCGTTTGCTCTTCTTCTTGCG (SEQ ID NO:7) . 1-1000 μg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravitreally. Treatment may be repeated as necessary until the infection is ablated.

EXAMPLE 13

A patient suffering from PKC-α-mediated cancer is treated with ISIS-3521, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence GTTCTCGCTGGTGAGTTTCA (SEQ ID NO:8) . 1-1000 μg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

EXAMPLE 14

A patient suffering from C-raf kinase-mediated cancer is treated with ISIS-5132, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence TCCCGCCTGTGACATGCATT (SEQ ID NO:9) . 1-1000 μg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

EXAMPLE 15

A patient suffering from C-raf kinase-mediated cancer is treated with ISIS-2503 (SEQ ID NO:10), ISIS-2570 (SEQ ID NO:ll) or ISIS-6957 (SEQ ID NO:12), each of which are synthesized according to the procedure of Example 3 or Example 19. 1-1000 μg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

Compounds 1, 2 and 3 of Examples 16, 17 and 18, respectively, are synthesized according to the procedure of Stec et al . [Nucleic Acids Res . , 19:5883 (1991)] and Stec and Lesnikowski [Methods in Molecular Biology, S. Agrawal, Ed., Volume 20, p. 285, 1993] .

EXAMPLE 16

SYNTHESIS OF 2-CHLORO-l,3,2-OXATHIAPHOSPHOLANE (1)

A mixture of pyridine (1 mol) , benzene (400 mL) , 2-mercaptoethanol (0.5 mol) and phosphorus trichloride (0.5 mol) are stirred at room temperature for 30 minutes. Pyridinium chloride is filtered off, solvent is evaporated under reduced pressure and crude product is purified by distillation under reduced pressure. The fraction boiling at 70-72°C/20 mm Hg is collected and characterized by 31P NMR.

EXAMPLE 17

SYNTHESIS OF N,N-DIISOPROPYLAMINO-1,3,2-OXATHIAPHOSPHOLANE (2) Compound 1 (0.2 mol) is dissolved in n-pentane

(300 mL) and diisopropylamine (0.4 mol) is added dropwise. The reaction mixture is stirred at room temperature for 30 minutes, after which diisopropylamine hydrochloride is filtered off, solvent is evaporated under reduced pressure and crude product is purified by vacuum distillation.

Product 2 is obtained as the fraction boiling at 70°C/0.1 mm Hg and is characterized by 31P NMR and mass spectroscopy.

EXAMPLE 18

SYNTHESIS OF 5' -0-DIMETHOXYTRITYLTHYMIDINE-3' -O[2-THIONO- 1,3,2-OXATHIAPHOPSHOLANE] (3)

5' -0-Dimthoxytritylthymidine (10 mmol) and 1H- tetrazole (11 mmol) are vacuum dried and dissolved in dichloromethane (25 mL) . Compound 2 (11 mmol) is added to the solution and the reaction mixture is stirred at room temperature for 2 hours. Dried elemental sulfur (15 mmol) is added and the reaction mixture is stirred and left at room temperature for 16 hours. Unreacted sulfur is then filtered off and the filtrate is concentrated under reduced pressure. The residue is dissolved in chloroform (3 mL) and purified by silica gel (230-400 mesh) column chromatography, eluting first with chloroform, and next with chloroform:methanol (97:3) . Individual diastereomeric species of compound 3 are obtained by column chromatography on silica gel. Compound 3 is dissolved in ethyl acetate and applied on a silica gel 60H column. Ethyl acetate is used as the eluting solvent, and elution is monitored by HPTLC (silica gel 60, ethyl acetate as the developing solvent) . Fractions containing separated diastereomers of compound 3 are concentrated under reduced pressure and the residue is characterized by 31P NMR and HPLC (Lichrospher SilOO, 5 μM, ethyl acetate as eluant, flow rate 3 mL/minute) . The fast eluting fraction corresponds to the Sp diastereomer, and the slow eluting fraction is the Rp diastereomer.

EXAMPLE 19 STEREOSPECIFIC CONTROL OF REACTION BETWEEN 5' -OH NUCLEOSIDES AND DIASTEREOMERICALLY PURE 3 IN SOLID PHASE AUTOMATED SYNTHESIS

The procedure of Stec et al . was followed using an Applied Biosystems (Foster City, CA) model 380B automated DNA synthesizer. The reaction between a 5' -OH nucleoside and diastereomerically pure nucleoside oxathiaphospholane, such as 3, requires the use of 1, 8-diazabicyclo (5.4.0)undec- 7-ene (DBU) as the catalyst. Because the commercially available linker used for the attachment of oligonucleotide to support matrix in automated DNA synthesis is unstable to DBU, a modification is required in the linker. A suitable linker for oligonucleotide synthesis via the oxathiaphospholane method is a "succinic-sarcosinyl" linker that is resistant to DBU, and can be hydrolyzed by concentrated ammonium hydroxide at room temperature in less than 1 hour.

(A) Synthesis of 5' -O-dimethoxytritylnucleosides bound to solid matrix via "succinic-sarcosinyl" linker:

(1) N-Fmoc-sarcosine (Bachem Bioscience, Inc., Philadelphia, PA) (1.6 mmol) is added to long chain alkyl amine-CPG (LCA-CPG, Sigma, St. Louis, MO) (2 g) and dried under vacuum. Anhydrous DMF (5 mL) , pyridine (0.5 mL) and DCC (2.4 mmol) are added and the reaction mixture is shaken at room temperature for 12 hours. The solvent is then filtered off and the support is washed with methanol :acetonitrile:pyridine (1:1:1, 3x20 mL) . The N-Fmoc protecting group is removed by treating the support with 10 mL of a 10% solution of piperidine in pyridine. N- sarcosinylated LCA-CPG is washed with methanol:acetonitrile:pyridine (1:1:1, 3x20 mL) and dried under vacuum.

(2) 5' -O-Dimethoxytritylnucleoside is added to the sarcosinylated LCA-CPG obtained as described in (1) in the presence of DMF (2 mL) , pyridine ( 0.2 mL) and DCC (50 mg) . The reaction mixture is shaken at room temperature for 12 hours and then washed with methanol:acetonitrile:pyridine (1:1:1, 3x20 mL) . After drying, the support is treated with N-methylimidazole:THF (1 mL) and acetic anhydride/lutidine (1 mL) for 15 minutes. The support is then washed with methanol:acetonitrile:pyridine (1:1:1, 3x10 mL) , followed by acetonitrile (3x10 mL) , and then dried under vacuum. (B) Diastereomerically pure activated nucleosides are subsequently added onto the oligonucleotide attached to the sarcosinyl LCA-CPG support in the presence of a 300-fold molar excess of DBU. The diastereomers of activated nucleosides are separated by column chromatography [silica gel 60H, ethyl acetate is used as the eluting solvent, elution is monitored by HPTLC (silica gel 60, ethyl acetate as the developing solvent)] prior to use in the coupling reaction. The synthetic protocol is shown in Table 3.

Table 3 Chemical steps for one synthesis cycle

Reagent or solvent Purpose Time

(minutes) Trichloroacetic acid in Detritylation 1.5 dichloromethane (2:98)

Acetonitrile Wash 2

Activated nucleoside (with Coupling 10

DBU) in acetonitrile Acetonitrile Wash 2

Acetic anhydride/lutidine Capping 1 in THF (1:1:8) and N-methylimidazole in THF (4:21) Acetonitrile Wash 1

The diastereomeric purity of the phosphorothioate oligonucleotide can be determined by 31P NMR, by HPLC (Lichrospher SilOO, 5 μM, ethyl acetate as eluant, flow rate 3 mL/minute) , enzymatically or by electrophoretic methods.

EXAMPLE 20

TREATMENT OF A DISEASE STATE IN A HUMAN PATIENT

The oligonucleotides of the invention may be used for treatment of various disease states. Treatment of a patient diagnosed with a particular disease state comprises administration of an effective dose of the olignucleotide, in a pharmaceutically accepted formulation, to the patient via an appropriate route. The effective oligonucleotide dose depends on the disease state being treated, the severity of the disease state and the age of the patient being treated. The effective dose of an oligonucleotide may be determined based on its IC50 and is a routine procedure for one of skill in the art. Alternatively, the effective dose of the oligomer may be determined by using the pharmacokinetics software program TopFit. For example, dosage of oligonucleotides may vary from 0.01 μg (for children) to 100 g (for adults) per kg of body weight depending on progression of the disease state. Similarly, the frequency of dosing depends on the progression of the disease state and may vary from once or more daily to once every 20 years.

The route of oligonucleotide administration depends on the disease state being treated. For example, administration of an oligonucleotide to a patient being treated for an inflammatory disorder may be accomplished either via oral or rectal routes. For treatment of a patient afflicted with AIDS, the most effective method of oligonucleotide administration may be an oral route or by subcutaneous injection. Cancers such as breast cancer may be treated via subcutaneous injection, while colon cancer may be treated via oral or rectal administration of the oligonucleotide. Diseases or disorders of the central nervous system may best be treated by intrathecal or intraventricular administration for delivery of the oligonucleotide to the spinal column or the brain of the patient.

Following oligonucleotide administration, the patient may be monitored for alleviation of symptoms associated with the disease state. Subsequently, the dosage may be adjusted (increased or decreased) depending upon the severity and amenability of the disease state to treatment. It may be preferable to administer oligonucleotides of the invention in combination with other traditional therapeutics. The oligonucleotides may be administered in combination with drugs including, but not limited to, AZT for the treatment of patients afflicted with AIDS, sulfasalazine for the treatment of an inflammatory disorder such as ulcerative colitis, and 5-fluorouracil for the treatment of colon cancer.

Also, it may be desirable to administer maintenance therapy to a patient who has been successfully treated for a disease state. The dosage and frequency of oligonucleotide administration as part of a maintenance regimen may vary from 0.01 μg to 100 g per kg of body weight, ranging from once or more daily to once every several years.

EXAMPLE 21

IN_ΕAv__^TRICULAR ADMINISTRATION OF OLIGONUCLEOTIDES Intraventricular drug administration, for the direct delivery of drug to the brain of a patient, may be desired for the treatment of patients with diseases afflicting the brain. To effect this mode of oligonucleotide administration, a silicon catheter is surgically introduced into a ventricle of the brain of a human patient, and is connected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis, MN) that has been surgically implanted in the abdominal region [Cancer Research, 44:1698 (1984)] . The pump is used to inject the oligonucleotides and allows precise dosage adjustments and variation in dosage schedules with the aid of an external programming device. The reservoir capacity of the pump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1 mL/h. Depending on the frequency of administration, ranging from daily to monthly, and the dose of drug to be administered, ranging from 0.01 μg to 100 g per kg of body weight, the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by percutaneous puncture of the self-sealing septum of the pump.

EXAMPLE 22

INTΕATHECAL ADMINISTRATION OF OLIGONUCLEOTIDES

Intrathecal drug administration for the introduction of drug into the spinal column of a patient may be desired for the treatment of patients with diseases of the central nervous system. To effect this route of oligonucleotide administration, a silicon catheter is surgically implanted into the L3-4 lumbar spinal interspace of a human patient, and is connected to a subcutaneous infusion pump which has been surgically implanted in the upper abdominal region [The Annals of Pharmaco therapy, 27:912 (1993) and Cancer, 41:1270 (1993] . The pump is used to inject the oligonucleotides and allows precise dosage adjustments and variations in dose schedules with the aid of an external programming device. The reservoir capacity of the pump is 18-20 mL, and infusion rates may vary from 0.1 mL/h to 1 mL/h. Depending on the frequency of drug administration, ranging from daily to monthly, and dosage of drug to be administered, ranging from 0.01 μg to 100 g per kg of body weight, the pump reservoir may be refilled at 3- 10 week intervals. Refilling of the pump is accomplished by a single percutaneous puncture to the self-sealing septum of the pump. EXAMPLE 23 A patient suffering from AIDS is treated with ISIS-

5320, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence TTGGGGTT (SEQ ID NO:17) . 1-1000 μg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravitreally. Treatment may be repeated as necessary until the infection is ablated.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: ISIS Pharmaceuticals, Inc.,et al.

(ii) TITLE OF INVENTION: Oligonucleotides Having Phosphorothioate Linkages Of High Chiral Purity

(iii) NUMBER OF SEQUENCES: 17

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz _ Norris

(B) STREET: One Liberty Place - 46th Floor

(C) CITY: Philadelphia

(D) STATE: PA

(E) COUNTRY: U.S.A.

(F) ZIP: 19103

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 inch disk, 720 Kb

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: WordPerfect 6.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US96/08757

(B) FILING DATE: 05-JUN-1996

(C) CLASSIFICATION: N/A

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:08/471, 967

(B) FILING DATE: 06-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/467,597

(B) FILING DATE: 06-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:08/468,447

(B) FILING DATE: 06-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:08/468,569

(B) FILING DATE: 06-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:08/466, 692

(B) FILING DATE: 06-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:08/471, 966

(B) FILING DATE: 06-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:08/469, 851

(B) FILING DATE: 06-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:08/470,129

(B) FILING DATE: 06-JUN-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Joseph Lucci

(B) REGISTRATION NUMBER: 33,307

(C) REFERENCE/DOCKET NUMBER: ISIS-2298

SUBSTITUTE SHEET (RULE 28) (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-568-3100

(B) TELEFAX: 215-568-3439

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

CCTTTCGCGA CCCAACACTA 20

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

GCCTTTCGCG ACCCAACACT 20

(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GTACCACAAG GCCTTTCGCG 20

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4:

GTGCTCATGG TGCACGGTCT 20

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5

TTTAGGATTC GTGCTCATGG 20

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

SUBSTITUTE SHEET (RULE 28) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GCCCAAGCTG GCATCCGTCA 20

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GCGTTTGCTC TTCTTCTTGC G 21

(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GTTCTCGCTG GTGAGTTTCA 20

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

TCCCGCCTGT GACATGCATT 20

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

TCCGTCATCG CTCCTCAGGG 20

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

CCACACCGAC GGCGCCC 17

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)' (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:12:

CAGTGCCTGC GCCGCGCTCG 20

(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

CGACTATGCA AGTAC 15

(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

GTACTTGCAT AGTCGATCGG AAAATAGGGT TCTCATCTCC CGGGATTTGG 50

TTTGAG 56

(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

CTCAACCAAA TCCCGGGAGA TGAGAACCCT ATTTTCCGAT C 41

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

GACTATGCAA GTAC 14

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

TTGGGGTT 8

Claims

WHAT IS CLAIMED IS:
1. An oligonucleotide represented by SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:17 wherein at least 75% of the nucleoside units are joined together by Sp phosphorothioate 3' to 5' linkages.
2. The oligonucleotide of claim 1 wherein all of the nucleoside units are joined together by Sp phosphorothioate 3' to 5' linkages.
3. A composition containing an oligonucleotide of claim 1 and an acceptable carrier.
4. An oligonucleotide represented by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:17 wherein at least 75% of the nucleoside units are joined together by Rp phosphorothioate 3' to 5' linkages.
5. The oligonucleotide of claim 4 wherein all of the nucleoside units are joined together by Rp phosphorothioate 3' to 5' linkages.
6. A composition containing an oligonucleotide of claim 4 and an acceptable carrier.
PCT/US1996/008757 1991-10-15 1996-06-05 Oligonucleotides having phosphorothioate linkages of high chiral purity WO1996039154A1 (en)

Priority Applications (16)

Application Number Priority Date Filing Date Title
US08/471,966 1995-06-06
US08/469,851 US5587361A (en) 1991-10-15 1995-06-06 Oligonucleotides having phosphorothioate linkages of high chiral purity
US08/471,966 US5661134A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating Ha-ras or Ki-ras having phosphorothioate linkages of high chiral purity
US08/467,597 1995-06-06
US08/468,447 US5576302A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating hepatitis C virus having phosphorothioate linkages of high chiral purity
US08/470,129 1995-06-06
US08/466,692 1995-06-06
US08/467,597 US5607923A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating cytomegalovirus having phosphorothioate linkages of high chiral purity
US08/468,447 1995-06-06
US08/469,851 1995-06-06
US08/468,569 US5620963A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating protein kinase C having phosphorothioate linkages of high chiral purity
US08/471,967 US5599797A (en) 1991-10-15 1995-06-06 Oligonucleotides having phosphorothioate linkages of high chiral purity
US08/468,569 1995-06-06
US08/470,129 US5635488A (en) 1991-10-15 1995-06-06 Compounds having phosphorodithioate linkages of high chiral purity
US08/471,967 1995-06-06
US08/466,692 US5654284A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating RAF kinase having phosphorothioate linkages of high chiral purity

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
JP50125497A JPH10510433A (en) 1995-06-06 1996-06-05 Oligonucleotides having phosphorothioate linkages of high chiral purity
AU62528/96A AU698739B2 (en) 1995-06-06 1996-06-05 Oligonucleotides having phosphorothioate linkages of high chiral purity
KR1019970709017A KR100257972B1 (en) 1995-06-06 1996-06-05 Oligonucleotides having phosphorothioate linkages of high chiral purity
EP96921270A EP0831854A4 (en) 1995-06-06 1996-06-05 Oligonucleotides having phosphorothioate linkages of high chiral purity
NO975558A NO975558L (en) 1995-06-06 1997-12-02 Oligonucleotides with fosfortioatbindinger high chiral purity

Publications (1)

Publication Number Publication Date
WO1996039154A1 true WO1996039154A1 (en) 1996-12-12

Family

ID=27575447

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/008757 WO1996039154A1 (en) 1991-10-15 1996-06-05 Oligonucleotides having phosphorothioate linkages of high chiral purity

Country Status (6)

Country Link
EP (1) EP0831854A4 (en)
JP (3) JPH10510433A (en)
AU (1) AU698739B2 (en)
CA (1) CA2223103A1 (en)
NO (1) NO975558L (en)
WO (1) WO1996039154A1 (en)

Cited By (117)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032589A1 (en) * 1996-03-07 1997-09-12 Novartis Ag Combinations for treatment of proliferative diseases
WO1999005160A2 (en) * 1997-07-25 1999-02-04 Hybridon, Inc. Oligonuclotides having 3' terminal stereospecific phosphorothioates
WO2000006588A1 (en) * 1998-07-27 2000-02-10 University Of Iowa Research Foundation STEREOISOMERS OF CpG OLIGONUCLEOTIDES AND RELATED METHODS
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
EP1097162A2 (en) * 1998-07-14 2001-05-09 Isis Pharmaceuticals, Inc. Oligonucleotides having site specific chiral phosphorothioate internucleoside linkages
WO2003089620A2 (en) 2002-04-19 2003-10-30 Diversa Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
WO2004090099A2 (en) 2003-04-04 2004-10-21 Diversa Corporation Pectate lyases, nucleic acids encoding them and methods for making and using them
WO2005021714A2 (en) 2003-08-11 2005-03-10 Diversa Corporation Laccases, nucleic acids encoding them and methods for making and using them
US6881831B2 (en) 2001-05-16 2005-04-19 Migenix Inc. Nucleic acid-based compounds and methods of use thereof
WO2006009676A2 (en) 2004-06-16 2006-01-26 Diversa Corporation Compositions and methods for enzymatic decolorization of chlorophyll
WO2006101584A2 (en) 2005-03-15 2006-09-28 Diversa Corporation Cellulases, nucleic acids encoding them and methods for making and using them
USRE39464E1 (en) * 1998-07-14 2007-01-09 Isis Pharmaceuticals Inc. Oligonucleolotides having site specific chiral phosphorothioate internucleoside linkages
WO2007022151A2 (en) 2005-08-15 2007-02-22 Vaxin, Inc. Immunization of avians by administration of non-replicating vectored vaccines
WO2007095398A2 (en) 2006-02-14 2007-08-23 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
WO2008036916A2 (en) 2006-09-21 2008-03-27 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2008036863A2 (en) 2006-09-21 2008-03-27 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
WO2008080093A2 (en) 2006-12-21 2008-07-03 Verenium Corporation Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
WO2008095033A2 (en) 2007-01-30 2008-08-07 Verenium Corporation Enzymes for the treatment of lignocellulosics, nucleic acids encoding them and methods for making and using them
EP1985630A2 (en) 2001-01-03 2008-10-29 Senomyx, Inc. T1R taste receptors and genes encoding same
EP2003451A2 (en) 2001-06-26 2008-12-17 Senomyx, Inc. T1R hetero-oligomeric taste receptors and cell lines that express said receptors and use thereof for identification of taste compounds
US7494805B2 (en) 2003-02-14 2009-02-24 Biogen Idec Ma Inc. Expression cassette and vector for transient or stable expression of exogenous molecules
WO2009045627A2 (en) 2007-10-03 2009-04-09 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
WO2009088949A1 (en) 2008-01-03 2009-07-16 Verenium Corporation Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them
EP2112227A1 (en) 2006-03-07 2009-10-28 Cargill, Incorporated Aldolases, nucleic acids encoding them and methods for making and using them
WO2010025395A2 (en) 2008-08-29 2010-03-04 Verenium Corporation Hydrolases, nucleic acids encoding them and methods for making and using them
WO2010031074A2 (en) 2008-09-15 2010-03-18 Genentech, Inc. Compositions and methods for regulating cell osmolarity
EP2194133A2 (en) 2003-03-06 2010-06-09 Verenium Corporation Amylases, nucleic acids encoding them and methods for making and using them
EP2216403A2 (en) 2006-02-02 2010-08-11 Verenium Corporation Esterases and related nucleic acids and methods
WO2010135588A2 (en) 2009-05-21 2010-11-25 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2011008768A1 (en) 2009-07-13 2011-01-20 Wayne State University Modified egfr ectodomain
US7888470B2 (en) 2003-08-06 2011-02-15 Senomyx, Inc. Chimeric T1R taste receptor polypeptides and nucleic acid sequences encoding and cell lines that express said chimeric T1R polypeptides
EP2298802A1 (en) 2000-03-07 2011-03-23 Senomyx, Inc. T1R3 taste receptors and genes encoding same
WO2011046812A1 (en) 2009-10-16 2011-04-21 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
WO2011046815A1 (en) 2009-10-16 2011-04-21 Bunge Oils, Inc. Oil degumming methods
WO2011048498A2 (en) 2009-10-19 2011-04-28 Stichting Het Nederlands Kanker Instituut Differentiation between brca2-associated tumours and sporadic tumours via array comparative genomic hybridization
WO2011048499A1 (en) 2009-10-19 2011-04-28 Stichting Het Nederlands Kanker Instituut Predicting response to anti-cancer therapy via array comparative genomic hybridization
WO2011048495A1 (en) 2009-10-19 2011-04-28 Stichting Het Nederlands Kanker Instituut Predicting benefit of anti-cancer therapy via array comparative genomic hybridization
US7994296B2 (en) 2002-03-27 2011-08-09 Perkinelmer Health Sciences, Inc. Arrays, computer program products and methods for in silico array-based comparative binding assays
EP2385108A1 (en) 2006-03-07 2011-11-09 Verenium Corporation Aldolases, nucleic acids encoding them and methods for making and using them
WO2011142818A1 (en) 2010-05-10 2011-11-17 Abbott Laboratories Detection of chromosomal abnormalities associated with endometrial cancer
EP2404930A1 (en) 2003-07-02 2012-01-11 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
EP2415864A1 (en) 2006-02-10 2012-02-08 Verenium Corporation Oligomerase-2 (or beta-xylosidase) enzymes, nucleic acids encoding them and methods for making and using them
WO2012038837A2 (en) 2010-09-20 2012-03-29 Stichting Het Nederlands Kanker Instituut Methods for predicting response to anti-cancer therapy in cancer patients
WO2012051055A2 (en) 2010-10-06 2012-04-19 Bp Corporation North America Inc. Variant cbh i polypeptides
EP2444413A1 (en) 2006-08-04 2012-04-25 Verenium Corporation Methods for oil or gas well drilling, washing and/or fracturing
EP2458016A1 (en) 2005-09-02 2012-05-30 The Regents of the University of California Methods and probe combinations for detecting melanoma
US8481286B2 (en) 2008-08-12 2013-07-09 Allylix, Inc. Method for production of isoprenoid compounds
WO2013124740A2 (en) 2012-02-23 2013-08-29 Stichting Vu-Vumc BRCA DEFICIENCY PROTEIN AND mRNA SIGNATURES USEFUL IN IDENTIFICATION OF PATIENTS WITH BRCA-DEFICIENT TUMORS AND PREDICTING BENEFIT OF ANTI-CANCER THERAPY IN CANCER PATIENTS
WO2013192545A1 (en) 2012-06-22 2013-12-27 The Regents Of The University Of California Compositions and methods for mediating plant stomatal development in response to carbon dioxide and applications for engineering drought tolerance in plants
WO2014012090A1 (en) 2012-07-13 2014-01-16 The Broad Institute, Inc. Molecular sleds comprising a positively-charged amino acid sequence and a molecular cargo and uses thereof
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
EP2706122A2 (en) 2008-01-03 2014-03-12 Verenium Corporation Isomerases, nucleic acids encoding them and methods for making and using them
WO2014093622A2 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
WO2014093655A2 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
WO2014093635A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
WO2014093709A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
WO2014093701A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof
WO2014093718A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Methods, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
US8821884B2 (en) 2004-07-27 2014-09-02 The Regents Of The University Of California Compositions and methods using MD-2 mutants and chimeric proteins
WO2014145599A2 (en) 2013-03-15 2014-09-18 The Broad Institute, Inc. Recombinant virus and preparations thereof
WO2014204728A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells
WO2014204729A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components
EP2821413A1 (en) 2011-11-30 2015-01-07 The Broad Institute, Inc. Nucleotide-specific recognition sequences for designer tal effectors
US8969254B2 (en) 2010-12-16 2015-03-03 Dana-Farber Cancer Institute, Inc. Oligonucleotide array for tissue typing
EP2853593A1 (en) 2003-03-07 2015-04-01 DSM IP Assets B.V. Hydrolases, nucleic acids encoding them and mehods for making and using them
EP2853269A1 (en) 2008-05-19 2015-04-01 Advaxis, Inc. Dual delivery system for heterologous antigens
WO2015057671A1 (en) 2013-10-14 2015-04-23 The Broad Institute, Inc. Artificial transcription factors comprising a sliding domain and uses thereof
US9017679B2 (en) 2005-08-30 2015-04-28 University Of Miami Immunomodulating tumor necrosis factor receptor 25 (TNFR25) agonists, antagonists, and immunotoxins
US9017660B2 (en) 2009-11-11 2015-04-28 Advaxis, Inc. Compositions and methods for prevention of escape mutation in the treatment of Her2/neu over-expressing tumors
WO2015089465A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
WO2015089351A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders
WO2015089364A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Crystal structure of a crispr-cas system, and uses thereof
WO2015089419A2 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using particle delivery components
EP2886658A1 (en) 2005-03-10 2015-06-24 BASF Enzymes LLC Lyase enzymes, nucleic acids encoding them and methods for making and using them
US9072313B2 (en) 2006-04-21 2015-07-07 Senomyx, Inc. Comestible compositions comprising high potency savory flavorants, and processes for producing them
US9084747B2 (en) 2009-11-11 2015-07-21 Advaxis, Inc. Compositions and methods for prevention of escape mutation in the treatment of HER2/NEU over-expressing tumors
WO2015127134A2 (en) 2014-02-20 2015-08-27 Allergan, Inc. Complement component c5 antibodies
WO2015130826A1 (en) 2014-02-27 2015-09-03 Allergan, Inc. COMPLEMENT FACTOR Bb ANTIBODIES
EP2959917A2 (en) 2007-10-19 2015-12-30 The Regents of The University of California Compositions and methods for ameliorating cns inflammation, psychosis, delirium, ptsd or ptss
US9226958B2 (en) 2010-10-01 2016-01-05 University Of Georgia Research Foundation, Inc. Use of Listeria vaccine vectors to reverse vaccine unresponsiveness in parasitically infected individuals
WO2016049251A1 (en) 2014-09-24 2016-03-31 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling mutations in leukocytes
WO2016049163A2 (en) 2014-09-24 2016-03-31 The Broad Institute Inc. Use and production of chd8+/- transgenic animals with behavioral phenotypes characteristic of autism spectrum disorder
EP3009511A2 (en) 2015-06-18 2016-04-20 The Broad Institute, Inc. Novel crispr enzymes and systems
WO2016094880A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Delivery, use and therapeutic applications of crispr systems and compositions for genome editing as to hematopoietic stem cells (hscs)
WO2016094872A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Dead guides for crispr transcription factors
WO2016094867A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Protected guide rnas (pgrnas)
WO2016094874A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Escorted and functionalized guides for crispr-cas systems
WO2016100974A1 (en) 2014-12-19 2016-06-23 The Broad Institute Inc. Unbiased identification of double-strand breaks and genomic rearrangement by genome-wide insert capture sequencing
WO2016106244A1 (en) 2014-12-24 2016-06-30 The Broad Institute Inc. Crispr having or associated with destabilization domains
WO2016106236A1 (en) 2014-12-23 2016-06-30 The Broad Institute Inc. Rna-targeting system
WO2016108926A1 (en) 2014-12-30 2016-07-07 The Broad Institute Inc. Crispr mediated in vivo modeling and genetic screening of tumor growth and metastasis
US9499627B2 (en) 2009-08-03 2016-11-22 University Of Miami Method for in vivo expansion of T regulatory cells
WO2016205764A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Novel crispr enzymes and systems
WO2016205749A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Novel crispr enzymes and systems
WO2016205745A2 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Cell sorting
WO2016205728A1 (en) 2015-06-17 2016-12-22 Massachusetts Institute Of Technology Crispr mediated recording of cellular events
US9603925B2 (en) 2013-01-09 2017-03-28 University Of Miami Compositions comprising TL1A-Ig fusion protein for the regulation of T regulatory cells, and methods for their use
WO2017070605A1 (en) 2015-10-22 2017-04-27 The Broad Institute Inc. Type vi-b crispr enzymes and systems
WO2017075294A1 (en) 2015-10-28 2017-05-04 The Board Institute Inc. Assays for massively combinatorial perturbation profiling and cellular circuit reconstruction
US9650639B2 (en) 2008-05-19 2017-05-16 Advaxis, Inc. Dual delivery system for heterologous antigens
WO2017106657A1 (en) 2015-12-18 2017-06-22 The Broad Institute Inc. Novel crispr enzymes and systems
EP3190182A1 (en) 2004-03-08 2017-07-12 DSM IP Assets B.V. Phospholipases, nucleic acids encoding them and methods for making and using them
EP3196310A1 (en) 2007-04-27 2017-07-26 The Regents of The University of California Plant co2 sensors, nucleic acids encoding them, and methods for making and using them
US9745631B2 (en) 2011-12-20 2017-08-29 Dana-Farber Cancer Institute, Inc. Methods for diagnosing and treating oncogenic kras-associated cancer
US9752196B2 (en) 2009-10-26 2017-09-05 Abbott Molecular Inc. Detection of chromosomal abnormalities associated with prognosis of non small cell lung cancer
WO2017184768A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017184786A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Cpf1 complexes with reduced indel activity
WO2017189308A1 (en) 2016-04-19 2017-11-02 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017219027A1 (en) 2016-06-17 2017-12-21 The Broad Institute Inc. Type vi crispr orthologs and systems
WO2018005873A1 (en) 2016-06-29 2018-01-04 The Broad Institute Inc. Crispr-cas systems having destabilization domain
US9907849B2 (en) 2014-07-18 2018-03-06 Advaxis, Inc. Combination of a PD-1 antagonist and a listeria-based vaccine for treating prostate cancer
US10016617B2 (en) 2009-11-11 2018-07-10 The Trustees Of The University Of Pennsylvania Combination immuno therapy and radiotherapy for the treatment of Her-2-positive cancers
US10058599B2 (en) 2012-03-12 2018-08-28 Advaxis, Inc. Suppressor cell function inhibition following Listeria vaccine treatment
US10064898B2 (en) 2011-03-11 2018-09-04 Advaxis, Inc. Listeria-based adjuvants
WO2018208910A1 (en) 2017-05-09 2018-11-15 The Broad Institute Inc. Gut microbiome function predicts response to anti-integrin biologic therapy in inflammatory bowel diseases
US10130590B2 (en) 2013-10-01 2018-11-20 Dana-Farber Cancer Institute, Inc. Methods of treating cancer with atovaquone-related compounds
US10138479B2 (en) 2012-05-24 2018-11-27 Dana-Farber Cancer Institute, Inc. Targeting the glutamine to pyruvate pathway for treatment of oncogenic Kras-associated cancer

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG171914A1 (en) 2008-12-02 2011-07-28 Chiralgen Ltd Method for the synthesis of phosphorus atom modified nucleic acids
MX342945B (en) 2009-07-06 2016-10-18 Ontorii Inc * Novel nucleic acid prodrugs and methods use thereof.
KR20140068884A (en) 2011-07-19 2014-06-09 웨이브 라이프 사이언시스 피티이. 리미티드 Methods for the synthesis of functionalized nucleic acids
WO2014012081A2 (en) 2012-07-13 2014-01-16 Ontorii, Inc. Chiral control
US9598458B2 (en) 2012-07-13 2017-03-21 Wave Life Sciences Japan, Inc. Asymmetric auxiliary group
JP6246121B2 (en) 2012-07-13 2017-12-13 株式会社新日本科学 Chiral-nucleic acid adjuvants
WO2015108047A1 (en) 2014-01-15 2015-07-23 株式会社新日本科学 Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
JPWO2015108046A1 (en) 2014-01-15 2017-03-23 株式会社新日本科学 Chiral acid adjuvants and an antiallergic agent having an anti-allergic action
EP3095459A4 (en) 2014-01-15 2017-08-23 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
SG11201605782UA (en) 2014-01-16 2016-08-30 Wave Life Sciences Ltd Chiral design

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5212295A (en) * 1990-01-11 1993-05-18 Isis Pharmaceuticals Monomers for preparation of oligonucleotides having chiral phosphorus linkages
US5506212A (en) * 1990-01-11 1996-04-09 Isis Pharmaceuticals, Inc. Oligonucleotides with substantially chirally pure phosphorothioate linkages

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006659A1 (en) * 1992-07-01 1995-03-09 Isis Pharmaceuticals, Inc. Amine-derivatized nucleosides and oligonucleosides
JP2693643B2 (en) * 1991-10-15 1997-12-24 アイシス・ファーマシューティカルス・インコーポレーテッド Oligonucleotide having a chiral phosphorus bond
AT230023T (en) * 1990-08-14 2003-01-15 Isis Pharmaceuticals Inc Manipulation of cell adhesion by oligonucleotides
WO1994008003A1 (en) * 1991-06-14 1994-04-14 Isis Pharmaceuticals, Inc. ANTISENSE OLIGONUCLEOTIDE INHIBITION OF THE ras GENE
US5681747A (en) * 1992-03-16 1997-10-28 Isis Pharmaceuticals, Inc. Nucleic acid sequences encoding protein kinase C and antisense inhibition of expression thereof
DE69330372T2 (en) * 1992-09-10 2002-03-14 Isis Pharmaceuticals Inc COMPOSITIONS AND METHODS FOR THE TREATMENT OF HEPATITIS C VIRUS WITH ASSOCIATED DISEASES

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5212295A (en) * 1990-01-11 1993-05-18 Isis Pharmaceuticals Monomers for preparation of oligonucleotides having chiral phosphorus linkages
US5506212A (en) * 1990-01-11 1996-04-09 Isis Pharmaceuticals, Inc. Oligonucleotides with substantially chirally pure phosphorothioate linkages
US5521302A (en) * 1990-01-11 1996-05-28 Isis Pharmaceuticals, Inc. Process for preparing oligonucleotides having chiral phosphorus linkages

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0831854A4 *

Cited By (190)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032589A1 (en) * 1996-03-07 1997-09-12 Novartis Ag Combinations for treatment of proliferative diseases
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
WO1999005160A2 (en) * 1997-07-25 1999-02-04 Hybridon, Inc. Oligonuclotides having 3' terminal stereospecific phosphorothioates
WO1999005160A3 (en) * 1997-07-25 1999-04-08 Hybridon Inc Oligonuclotides having 3' terminal stereospecific phosphorothioates
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
EP1097162A2 (en) * 1998-07-14 2001-05-09 Isis Pharmaceuticals, Inc. Oligonucleotides having site specific chiral phosphorothioate internucleoside linkages
EP1097162A4 (en) * 1998-07-14 2002-03-06 Isis Pharmaceuticals Inc Oligonucleotides having site specific chiral phosphorothioate internucleoside linkages
US6440943B1 (en) 1998-07-14 2002-08-27 Isis Pharmaceuticals, Inc. Oligonucleotides having site specific chiral phosphorothioate internucleoside linkages
USRE39464E1 (en) * 1998-07-14 2007-01-09 Isis Pharmaceuticals Inc. Oligonucleolotides having site specific chiral phosphorothioate internucleoside linkages
WO2000006588A1 (en) * 1998-07-27 2000-02-10 University Of Iowa Research Foundation STEREOISOMERS OF CpG OLIGONUCLEOTIDES AND RELATED METHODS
EP2298802A1 (en) 2000-03-07 2011-03-23 Senomyx, Inc. T1R3 taste receptors and genes encoding same
EP1985630A2 (en) 2001-01-03 2008-10-29 Senomyx, Inc. T1R taste receptors and genes encoding same
GB2392157B (en) * 2001-05-16 2005-12-21 Micrologix Biotech Inc Nucleic acid-based compounds and methods of use thereof
US7709449B2 (en) 2001-05-16 2010-05-04 Migenix, Inc. Nucleic acid-based compounds and methods of use thereof
US6881831B2 (en) 2001-05-16 2005-04-19 Migenix Inc. Nucleic acid-based compounds and methods of use thereof
US7256179B2 (en) 2001-05-16 2007-08-14 Migenix, Inc. Nucleic acid-based compounds and methods of use thereof
EP2327985A2 (en) 2001-06-26 2011-06-01 Senomyx, Inc. T1R Hetero-oligomeric taste receptors and cell lines that express said receptors and use thereof for identification of taste compounds
EP2003451A2 (en) 2001-06-26 2008-12-17 Senomyx, Inc. T1R hetero-oligomeric taste receptors and cell lines that express said receptors and use thereof for identification of taste compounds
EP2293067A2 (en) 2001-06-26 2011-03-09 Senomyx, Inc. T1R hetero-oligomeric taste receptors and cell lines that express said receptors and use thereof for identification of taste compounds
US7994296B2 (en) 2002-03-27 2011-08-09 Perkinelmer Health Sciences, Inc. Arrays, computer program products and methods for in silico array-based comparative binding assays
WO2003089620A2 (en) 2002-04-19 2003-10-30 Diversa Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
EP2298871A1 (en) 2002-04-19 2011-03-23 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
US7494805B2 (en) 2003-02-14 2009-02-24 Biogen Idec Ma Inc. Expression cassette and vector for transient or stable expression of exogenous molecules
EP3023498A1 (en) 2003-03-06 2016-05-25 BASF Enzymes LLC Amylases, nucleic acids encoding them and methods for making and using them
EP2194133A2 (en) 2003-03-06 2010-06-09 Verenium Corporation Amylases, nucleic acids encoding them and methods for making and using them
EP2853593A1 (en) 2003-03-07 2015-04-01 DSM IP Assets B.V. Hydrolases, nucleic acids encoding them and mehods for making and using them
EP2341136A1 (en) 2003-04-04 2011-07-06 Verenium Corporation Pectate lyases, Nucleic Acids encoding them and methods for making and using them
WO2004090099A2 (en) 2003-04-04 2004-10-21 Diversa Corporation Pectate lyases, nucleic acids encoding them and methods for making and using them
EP2404930A1 (en) 2003-07-02 2012-01-11 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
EP2404929A1 (en) 2003-07-02 2012-01-11 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
EP2404931A1 (en) 2003-07-02 2012-01-11 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
EP2404928A1 (en) 2003-07-02 2012-01-11 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
EP2409981A1 (en) 2003-07-02 2012-01-25 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
US8895050B2 (en) 2003-08-06 2014-11-25 Senomyx, Inc. Flavors, flavor modifiers, tastants, taste enhancers, umami or sweet tastants, and/or enhancers and use thereof
US8404455B2 (en) 2003-08-06 2013-03-26 Senomyx, Inc. Chimeric T1R taste receptor polypeptides and nucleic acid sequences encoding and cell lines that express said chimeric T1R polypeptides
US9459250B2 (en) 2003-08-06 2016-10-04 Senomyx, Inc. Use of T1R3 venus flytrap region polypeptide to screen for taste modulators
US7888470B2 (en) 2003-08-06 2011-02-15 Senomyx, Inc. Chimeric T1R taste receptor polypeptides and nucleic acid sequences encoding and cell lines that express said chimeric T1R polypeptides
US9091686B2 (en) 2003-08-06 2015-07-28 Senomyx, Inc. Chimeric T1R taste receptor polypeptides and nucleic acid sequences encoding and cell lines that express said chimeric T1R polypeptides
US10060909B2 (en) 2003-08-06 2018-08-28 Senomyx, Inc. Flavors, flavor modifiers, tastants, taste enhancers, umami or sweet tastants, and/or enhancers and use thereof
WO2005021714A2 (en) 2003-08-11 2005-03-10 Diversa Corporation Laccases, nucleic acids encoding them and methods for making and using them
EP3190182A1 (en) 2004-03-08 2017-07-12 DSM IP Assets B.V. Phospholipases, nucleic acids encoding them and methods for making and using them
EP2468853A1 (en) 2004-06-16 2012-06-27 Verenium Corporation Composition and methods for enzymatic decolorization of chlorophyll
WO2006009676A2 (en) 2004-06-16 2006-01-26 Diversa Corporation Compositions and methods for enzymatic decolorization of chlorophyll
US8821884B2 (en) 2004-07-27 2014-09-02 The Regents Of The University Of California Compositions and methods using MD-2 mutants and chimeric proteins
EP2886658A1 (en) 2005-03-10 2015-06-24 BASF Enzymes LLC Lyase enzymes, nucleic acids encoding them and methods for making and using them
WO2006101584A2 (en) 2005-03-15 2006-09-28 Diversa Corporation Cellulases, nucleic acids encoding them and methods for making and using them
EP2949756A2 (en) 2005-03-15 2015-12-02 BP Corporation North America Inc. Cellulases, nucleic acids encoding them and methods for making and using them
WO2007022151A2 (en) 2005-08-15 2007-02-22 Vaxin, Inc. Immunization of avians by administration of non-replicating vectored vaccines
US9017679B2 (en) 2005-08-30 2015-04-28 University Of Miami Immunomodulating tumor necrosis factor receptor 25 (TNFR25) agonists, antagonists, and immunotoxins
US9839670B2 (en) 2005-08-30 2017-12-12 University Of Miami Immunomodulating tumor necrosis factor receptor 25 (TNFR25) agonists, antagonists, and immunotoxins
EP2458016A1 (en) 2005-09-02 2012-05-30 The Regents of the University of California Methods and probe combinations for detecting melanoma
EP2216403A2 (en) 2006-02-02 2010-08-11 Verenium Corporation Esterases and related nucleic acids and methods
EP2444487A1 (en) 2006-02-10 2012-04-25 Verenium Corporation Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them
EP2444489A1 (en) 2006-02-10 2012-04-25 Verenium Corporation Cellucloytic enzymes, nucleic acids encoding them and methods for making and using them
EP2444488A1 (en) 2006-02-10 2012-04-25 Verenium Corporation Cellucloytic enzymes, nucleic acids encoding them and methods for making and using them
EP2444490A1 (en) 2006-02-10 2012-04-25 Verenium Corporation Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them
EP2450439A1 (en) 2006-02-10 2012-05-09 Verenium Corporation Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them
EP2420570A1 (en) 2006-02-10 2012-02-22 Verenium Corporation Arabinofuranosidase enzymes, nucleic acids encoding them and methods for making and using them
EP2415864A1 (en) 2006-02-10 2012-02-08 Verenium Corporation Oligomerase-2 (or beta-xylosidase) enzymes, nucleic acids encoding them and methods for making and using them
EP2447363A1 (en) 2006-02-10 2012-05-02 Verenium Corporation Cellucloytic enzymes, nucleic acids encoding them and methods for making and using them
EP2548954A1 (en) 2006-02-14 2013-01-23 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
EP2548956A1 (en) 2006-02-14 2013-01-23 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
WO2007095398A2 (en) 2006-02-14 2007-08-23 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
EP2548955A1 (en) 2006-02-14 2013-01-23 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
EP2388316A2 (en) 2006-03-07 2011-11-23 Verenium Corporation Aldolases, nucleic acids encoding them and methods for making and using them
EP2385108A1 (en) 2006-03-07 2011-11-09 Verenium Corporation Aldolases, nucleic acids encoding them and methods for making and using them
EP2112227A1 (en) 2006-03-07 2009-10-28 Cargill, Incorporated Aldolases, nucleic acids encoding them and methods for making and using them
EP2322643A1 (en) 2006-03-07 2011-05-18 Cargill, Incorporated Aldolases, nucleic acids encoding them and methods for making and using them
EP3153580A2 (en) 2006-03-07 2017-04-12 BASF Enzymes LLC Aldolases, nucleic acids encoding them and methods for making and using them
EP2316962A1 (en) 2006-03-07 2011-05-04 Cargill, Incorporated Aldolases, nucleic acids encoding them and methods for making and using them
US9072313B2 (en) 2006-04-21 2015-07-07 Senomyx, Inc. Comestible compositions comprising high potency savory flavorants, and processes for producing them
EP2444413A1 (en) 2006-08-04 2012-04-25 Verenium Corporation Methods for oil or gas well drilling, washing and/or fracturing
EP2617816A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2617820A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2617728A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2008036863A2 (en) 2006-09-21 2008-03-27 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
EP2617819A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2617815A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2617729A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2397486A1 (en) 2006-09-21 2011-12-21 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2617821A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2617817A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2617823A2 (en) 2006-09-21 2013-07-24 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2008036916A2 (en) 2006-09-21 2008-03-27 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2620495A2 (en) 2006-09-21 2013-07-31 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2008080093A2 (en) 2006-12-21 2008-07-03 Verenium Corporation Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
EP2479266A1 (en) 2006-12-21 2012-07-25 Verenium Corporation Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
EP2479267A1 (en) 2006-12-21 2012-07-25 Verenium Corporation Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
EP3101128A1 (en) 2006-12-21 2016-12-07 BASF Enzymes LLC Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
WO2008095033A2 (en) 2007-01-30 2008-08-07 Verenium Corporation Enzymes for the treatment of lignocellulosics, nucleic acids encoding them and methods for making and using them
EP3196310A1 (en) 2007-04-27 2017-07-26 The Regents of The University of California Plant co2 sensors, nucleic acids encoding them, and methods for making and using them
WO2009045627A2 (en) 2007-10-03 2009-04-09 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
EP2708602A2 (en) 2007-10-03 2014-03-19 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
EP2959917A2 (en) 2007-10-19 2015-12-30 The Regents of The University of California Compositions and methods for ameliorating cns inflammation, psychosis, delirium, ptsd or ptss
EP2706122A2 (en) 2008-01-03 2014-03-12 Verenium Corporation Isomerases, nucleic acids encoding them and methods for making and using them
WO2009088949A1 (en) 2008-01-03 2009-07-16 Verenium Corporation Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them
EP2865750A2 (en) 2008-01-03 2015-04-29 BASF Enzymes LLC Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them
US9650639B2 (en) 2008-05-19 2017-05-16 Advaxis, Inc. Dual delivery system for heterologous antigens
EP2853269A1 (en) 2008-05-19 2015-04-01 Advaxis, Inc. Dual delivery system for heterologous antigens
US9644212B2 (en) 2008-05-19 2017-05-09 Advaxis, Inc. Dual delivery system for heterologous antigens
US8609371B2 (en) 2008-08-12 2013-12-17 Allylix, Inc. Isoprenoid compounds
US8481286B2 (en) 2008-08-12 2013-07-09 Allylix, Inc. Method for production of isoprenoid compounds
US8753842B2 (en) 2008-08-12 2014-06-17 Allylix, Inc. Method for production of isoprenoid compounds
WO2010025395A2 (en) 2008-08-29 2010-03-04 Verenium Corporation Hydrolases, nucleic acids encoding them and methods for making and using them
WO2010031074A2 (en) 2008-09-15 2010-03-18 Genentech, Inc. Compositions and methods for regulating cell osmolarity
WO2010135588A2 (en) 2009-05-21 2010-11-25 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2698374A1 (en) 2009-05-21 2014-02-19 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2011008768A1 (en) 2009-07-13 2011-01-20 Wayne State University Modified egfr ectodomain
US9499627B2 (en) 2009-08-03 2016-11-22 University Of Miami Method for in vivo expansion of T regulatory cells
US9512382B2 (en) 2009-10-16 2016-12-06 Bunge Global Innovation, Llc Oil degumming methods
WO2011046815A1 (en) 2009-10-16 2011-04-21 Bunge Oils, Inc. Oil degumming methods
WO2011046812A1 (en) 2009-10-16 2011-04-21 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
US9045712B2 (en) 2009-10-16 2015-06-02 Bunge Global Innovation, Llc Oil degumming methods
WO2011048495A1 (en) 2009-10-19 2011-04-28 Stichting Het Nederlands Kanker Instituut Predicting benefit of anti-cancer therapy via array comparative genomic hybridization
WO2011048499A1 (en) 2009-10-19 2011-04-28 Stichting Het Nederlands Kanker Instituut Predicting response to anti-cancer therapy via array comparative genomic hybridization
WO2011048498A2 (en) 2009-10-19 2011-04-28 Stichting Het Nederlands Kanker Instituut Differentiation between brca2-associated tumours and sporadic tumours via array comparative genomic hybridization
EP3272884A2 (en) 2009-10-26 2018-01-24 Abbott Molecular Inc. Detection of chromosomal abnormalities associated with prognosis of non small cell lung cancer
US9752196B2 (en) 2009-10-26 2017-09-05 Abbott Molecular Inc. Detection of chromosomal abnormalities associated with prognosis of non small cell lung cancer
US9084747B2 (en) 2009-11-11 2015-07-21 Advaxis, Inc. Compositions and methods for prevention of escape mutation in the treatment of HER2/NEU over-expressing tumors
US10016617B2 (en) 2009-11-11 2018-07-10 The Trustees Of The University Of Pennsylvania Combination immuno therapy and radiotherapy for the treatment of Her-2-positive cancers
US9017660B2 (en) 2009-11-11 2015-04-28 Advaxis, Inc. Compositions and methods for prevention of escape mutation in the treatment of Her2/neu over-expressing tumors
WO2011142818A1 (en) 2010-05-10 2011-11-17 Abbott Laboratories Detection of chromosomal abnormalities associated with endometrial cancer
WO2012038837A2 (en) 2010-09-20 2012-03-29 Stichting Het Nederlands Kanker Instituut Methods for predicting response to anti-cancer therapy in cancer patients
US9226958B2 (en) 2010-10-01 2016-01-05 University Of Georgia Research Foundation, Inc. Use of Listeria vaccine vectors to reverse vaccine unresponsiveness in parasitically infected individuals
US9943590B2 (en) 2010-10-01 2018-04-17 The Trustees Of The University Of Pennsylvania Use of Listeria vaccine vectors to reverse vaccine unresponsiveness in parasitically infected individuals
WO2012051055A2 (en) 2010-10-06 2012-04-19 Bp Corporation North America Inc. Variant cbh i polypeptides
US8969254B2 (en) 2010-12-16 2015-03-03 Dana-Farber Cancer Institute, Inc. Oligonucleotide array for tissue typing
US9463227B2 (en) 2011-03-11 2016-10-11 Advaxis, Inc. Listeria-based adjuvants
US10064898B2 (en) 2011-03-11 2018-09-04 Advaxis, Inc. Listeria-based adjuvants
EP2821413A1 (en) 2011-11-30 2015-01-07 The Broad Institute, Inc. Nucleotide-specific recognition sequences for designer tal effectors
EP2821505A2 (en) 2011-11-30 2015-01-07 The Broad Institute, Inc. Nucleotide-specific recognition sequences for designer tal effectors
US9745631B2 (en) 2011-12-20 2017-08-29 Dana-Farber Cancer Institute, Inc. Methods for diagnosing and treating oncogenic kras-associated cancer
WO2013124740A2 (en) 2012-02-23 2013-08-29 Stichting Vu-Vumc BRCA DEFICIENCY PROTEIN AND mRNA SIGNATURES USEFUL IN IDENTIFICATION OF PATIENTS WITH BRCA-DEFICIENT TUMORS AND PREDICTING BENEFIT OF ANTI-CANCER THERAPY IN CANCER PATIENTS
US10058599B2 (en) 2012-03-12 2018-08-28 Advaxis, Inc. Suppressor cell function inhibition following Listeria vaccine treatment
US10138479B2 (en) 2012-05-24 2018-11-27 Dana-Farber Cancer Institute, Inc. Targeting the glutamine to pyruvate pathway for treatment of oncogenic Kras-associated cancer
WO2013192545A1 (en) 2012-06-22 2013-12-27 The Regents Of The University Of California Compositions and methods for mediating plant stomatal development in response to carbon dioxide and applications for engineering drought tolerance in plants
WO2014012090A1 (en) 2012-07-13 2014-01-16 The Broad Institute, Inc. Molecular sleds comprising a positively-charged amino acid sequence and a molecular cargo and uses thereof
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
EP3494997A1 (en) 2012-07-25 2019-06-12 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
WO2014093635A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
WO2014093709A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
WO2014093701A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof
EP3434776A1 (en) 2012-12-12 2019-01-30 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
EP3327127A1 (en) 2012-12-12 2018-05-30 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
WO2014093622A2 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
WO2014093718A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Methods, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
EP3045537A1 (en) 2012-12-12 2016-07-20 The Broad Institute, Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
EP3064585A1 (en) 2012-12-12 2016-09-07 The Broad Institute, Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
WO2014093655A2 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US9603925B2 (en) 2013-01-09 2017-03-28 University Of Miami Compositions comprising TL1A-Ig fusion protein for the regulation of T regulatory cells, and methods for their use
WO2014145599A2 (en) 2013-03-15 2014-09-18 The Broad Institute, Inc. Recombinant virus and preparations thereof
WO2014204729A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components
WO2014204728A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells
US10130590B2 (en) 2013-10-01 2018-11-20 Dana-Farber Cancer Institute, Inc. Methods of treating cancer with atovaquone-related compounds
WO2015057671A1 (en) 2013-10-14 2015-04-23 The Broad Institute, Inc. Artificial transcription factors comprising a sliding domain and uses thereof
WO2015089364A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Crystal structure of a crispr-cas system, and uses thereof
WO2015089465A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
WO2015089351A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders
WO2015089419A2 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using particle delivery components
WO2015089354A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders
US9701743B2 (en) 2014-02-20 2017-07-11 Allergan, Inc. Complement component C5 antibodies
US9932395B2 (en) 2014-02-20 2018-04-03 Allergan, Inc. Nucleic acids encoding complement component C5 antibodies
WO2015127134A2 (en) 2014-02-20 2015-08-27 Allergan, Inc. Complement component c5 antibodies
WO2015130826A1 (en) 2014-02-27 2015-09-03 Allergan, Inc. COMPLEMENT FACTOR Bb ANTIBODIES
US9907849B2 (en) 2014-07-18 2018-03-06 Advaxis, Inc. Combination of a PD-1 antagonist and a listeria-based vaccine for treating prostate cancer
WO2016049163A2 (en) 2014-09-24 2016-03-31 The Broad Institute Inc. Use and production of chd8+/- transgenic animals with behavioral phenotypes characteristic of autism spectrum disorder
WO2016049251A1 (en) 2014-09-24 2016-03-31 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling mutations in leukocytes
WO2016094874A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Escorted and functionalized guides for crispr-cas systems
WO2016094880A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Delivery, use and therapeutic applications of crispr systems and compositions for genome editing as to hematopoietic stem cells (hscs)
WO2016094867A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Protected guide rnas (pgrnas)
WO2016094872A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Dead guides for crispr transcription factors
WO2016100974A1 (en) 2014-12-19 2016-06-23 The Broad Institute Inc. Unbiased identification of double-strand breaks and genomic rearrangement by genome-wide insert capture sequencing
WO2016106236A1 (en) 2014-12-23 2016-06-30 The Broad Institute Inc. Rna-targeting system
WO2016106244A1 (en) 2014-12-24 2016-06-30 The Broad Institute Inc. Crispr having or associated with destabilization domains
WO2016108926A1 (en) 2014-12-30 2016-07-07 The Broad Institute Inc. Crispr mediated in vivo modeling and genetic screening of tumor growth and metastasis
WO2016205728A1 (en) 2015-06-17 2016-12-22 Massachusetts Institute Of Technology Crispr mediated recording of cellular events
EP3009511A2 (en) 2015-06-18 2016-04-20 The Broad Institute, Inc. Novel crispr enzymes and systems
WO2016205749A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Novel crispr enzymes and systems
WO2016205764A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Novel crispr enzymes and systems
WO2016205711A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Novel crispr enzymes and systems
WO2016205745A2 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Cell sorting
WO2017070605A1 (en) 2015-10-22 2017-04-27 The Broad Institute Inc. Type vi-b crispr enzymes and systems
WO2017075294A1 (en) 2015-10-28 2017-05-04 The Board Institute Inc. Assays for massively combinatorial perturbation profiling and cellular circuit reconstruction
WO2017106657A1 (en) 2015-12-18 2017-06-22 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017184768A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017189308A1 (en) 2016-04-19 2017-11-02 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017184786A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Cpf1 complexes with reduced indel activity
WO2017219027A1 (en) 2016-06-17 2017-12-21 The Broad Institute Inc. Type vi crispr orthologs and systems
WO2018005873A1 (en) 2016-06-29 2018-01-04 The Broad Institute Inc. Crispr-cas systems having destabilization domain
WO2018208910A1 (en) 2017-05-09 2018-11-15 The Broad Institute Inc. Gut microbiome function predicts response to anti-integrin biologic therapy in inflammatory bowel diseases

Also Published As

Publication number Publication date
EP0831854A1 (en) 1998-04-01
NO975558L (en) 1998-01-26
JP2001114798A (en) 2001-04-24
NO975558D0 (en) 1997-12-02
AU698739B2 (en) 1998-11-05
CA2223103A1 (en) 1996-12-12
AU6252896A (en) 1996-12-24
JPH10510433A (en) 1998-10-13
JP2001103987A (en) 2001-04-17
EP0831854A4 (en) 2001-01-24

Similar Documents

Publication Publication Date Title
Lesnik et al. Oligodeoxynucleotides containing 2'-O-modified adenosine: synthesis and effects on stability of DNA: RNA duplexes
Shea et al. Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotide conjugates
Furdon et al. RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds
Hoke et al. Effects of phosphorothioate capping on antivense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection
US5808027A (en) N-2 substituted purines in oligonucleotides
CA2160016C (en) Method of forming oligonucleotides
JP2818031B2 (en) Oligonucleotides having conservative g ▲ under 4 ▼ core sequence
US6235886B1 (en) Methods of synthesis and use
EP1097162B1 (en) Oligonucleotides having site specific chiral phosphorothioate internucleoside linkages
US6506888B1 (en) 2′-O-amino-containing nucleoside analogs and polynucleotides
US6239272B1 (en) 2&#39;-O-alkylthioalkyl and 2&#39;-c-alkylthioalkyl-containing nucleic acids
US5491133A (en) Methods for blocking the expression of specifically targeted genes
CA2246229C (en) Sugar-modified gapped oligonucleotides
EP0734391B1 (en) Pna-dna-pna chimeric macromolecules
US6140491A (en) Nucleozymes
Vasseur et al. Oligonucleosides: Synthesis of a novel methylhydroxylamine-linked nucleoside dimer and its incorporation into antisense sequences
Giannaris et al. Oligoribonucleotides containing 2′, 5′-phosphodiester linkages exhibit binding selectivity for 3′, 5′-RNA over 3′, 5′-ssDNA
AU767646B2 (en) Oligonucleotide N3&#39;-P5&#39; thiophosphoramidates: their synthesis and use
US6737520B2 (en) Oligonucleotides having A-DNA form and B-DNA form conformational geometry
Sørensen et al. α-L-ribo-configured locked nucleic acid (α-L-LNA): synthesis and properties
Miller Oligonucleoside methylphosphonates as antisense reagents
JP5006485B2 (en) Oligoribonucleotides and ribonuclease to cut the Rna
US6500945B2 (en) Nucleotides having chiral phosphorus linkages
US6028188A (en) Synthetic oligomers having chirally pure phosphonate internucleosidyl linkages mixed with non-phosphonate internucleosidyl linkages
US5693773A (en) Triplex-forming antisense oligonucleotides having abasic linkers targeting nucleic acids comprising mixed sequences of purines and pyrimidines

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US US US US US US US US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase in:

Ref country code: CA

Ref document number: 2223103

Kind code of ref document: A

Format of ref document f/p: F

Ref document number: 2223103

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1019970709017

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 1996921270

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996921270

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1019970709017

Country of ref document: KR

WWG Wipo information: grant in national office

Ref document number: 1019970709017

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1996921270

Country of ref document: EP