WO2019040590A1 - Modulation de l'expression de fas soluble - Google Patents

Modulation de l'expression de fas soluble Download PDF

Info

Publication number
WO2019040590A1
WO2019040590A1 PCT/US2018/047473 US2018047473W WO2019040590A1 WO 2019040590 A1 WO2019040590 A1 WO 2019040590A1 US 2018047473 W US2018047473 W US 2018047473W WO 2019040590 A1 WO2019040590 A1 WO 2019040590A1
Authority
WO
WIPO (PCT)
Prior art keywords
dts
das
oligonucleotide
dgs
dcs
Prior art date
Application number
PCT/US2018/047473
Other languages
English (en)
Inventor
Caroline WOO
Original Assignee
Translate Bio Ma, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Translate Bio Ma, Inc. filed Critical Translate Bio Ma, Inc.
Publication of WO2019040590A1 publication Critical patent/WO2019040590A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-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
    • C12N15/1138—Non-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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the invention relates in part to compositions and methods for modulating gene expression, e.g., in the context of cell based therapies.
  • Adoptive cell transfer is a therapy that generally involves the transfer of cells into a subject for modulating one or more biological responses or functions in the subject.
  • Cells used in an adoptive cell transfer made be obtained from the patient (autologous cells) or from another individual (allogenic cells).
  • cells derived from the immune system of a subject e.g., monocytes, T cells, etc.
  • T cells may be obtained from the subject, modified (e.g., to express an engineered receptor) and/or expanded in culture and returned to the same subject.
  • T cell populations prepared according to methods provided herein are useful for cancer treatment via adoptive transfer, which may result in cancer regression or remission in subjects receiving the transfer.
  • T cells are prepared for adoptive transfer such that the make-up of the T cell population is controlled in order to optimize efficacy of treatment.
  • methods are provided for controlling the make-up of a T cell population (e.g., a CAR T cell population) in order to maintain or increase the percentage of naive T cells in the T cell population.
  • methods for adoptive transfer of specific T cell populations containing naive CD8-positive T cells are provided. Further aspects of the disclosure generally relate to methods for producing and/or maintaining cell populations containing naive CD8-positive T cells. In some embodiments,
  • oligonucleotides e.g., gapmers
  • other molecules are utilized to modulate the expression of genes that control the differentiation state of T cells, particularly naive CD8-positive T cells.
  • a method of maintaining or increasing the number of naive T cells in a T cell population comprising delivering ex vivo an oligonucleotide that inhibits the interaction of FAS-AS 1 with RBM5 to a T cell population comprising naive T cells.
  • delivering the oligonucleotide results in an increase in soluble Fas (sFas) expression in the naive T cells in the T cell population compared to sFas expression in control naive T cells in a control T cell population to which the oligonucleotide has not been delivered.
  • sFas soluble Fas
  • the T cell population is a CD4 + T cell population.
  • the method further comprises isolating T cells from a sample obtained from a donor subject; and selecting CD4 + T cells from the isolated T cells, thereby producing the T cell population comprising naive T cells to which the oligonucleotide is delivered.
  • the method further comprises administering the T cell population to a host subject after the oligonucleotide has been delivered to the T cell population.
  • the donor subject and the host subject are the same. In some embodiments, the donor subject and the host subject are different.
  • the method further comprises transfecting the T cell population with an expression construct encoding a chimeric antigen receptor (CAR).
  • the transfection occurs before delivery of the oligonucleotide to the T cell population. In some embodiments, the transfection occurs after delivery of the CAR.
  • the CAR is specific for a tumor antigen.
  • the host subject has cancer.
  • the T cell population is a human T cell population.
  • the oligonucleotide comprises a region of complementarity that is complementary with at least 8 nucleotides of FAS-AS 1. In some embodiments, the oligonucleotide reduces the level of FAS -AS 1. In some embodiments, the oligonucleotide is a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation of FAS -AS 1. In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 2 to 1833.
  • the oligonucleotide sterically interferes with the interaction of FAS-AS 1 with RBM5.
  • the oligonucleotide is single stranded.
  • the oligonucleotide is a mixmer. In some embodiments, the
  • oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 2 to 1833.
  • the oligonucleotide is a gapmer comprising a region of complementarity that is complementary with at least 8 nucleotides of FAS -AS 1.
  • the gapmer comprises the general formula:
  • each instance of X , X 3 is independently a modified or unmodified nucleotide, wherein m and o are independently integers in a range of 1 to 10, reflecting the number of instances of X 1 and X 3 , respectively, linked consecutively together through internucleotide linkages, wherein each instance of X 2 is a deoxyribonucleotide, wherein n is an integer in a range of 6 to 20, reflecting the number of instances of X 2 linked consecutively together through internucleotide linkages.
  • at least one of X 1 , X 3 is a 2'- modified nucleotide.
  • the 2'-modified nucleotide is a 2'-0,4'-C- bridged nucleotide. In some embodiments, 2'-modified nucleotide is a 2'-0,4'-C- methylene bridged nucleotide. In some embodiments, the a 2'-modified nucleotide is a 2'-0-methyl nucleotide. In some embodiments, the oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence set forth in Table 1 or Table 3 (e.g., a sequence selected from SEQ ID NOs: 2 to 1833).
  • each instance of X 1 , X 3 is a LNA nucleotide and each of the nucleotides in the oligonucleotide are linked by phosphorothioate linkages.
  • each instance of X 1 ⁇ 3 is a LNA nucleotide, m and o are each 3, n is 9, and each of the nucleotides in the oligonucleotide are linked by phosphorothioate linkages.
  • any cytosine LNAs in the oligonucleotide are 5-methylcytosine LNAs.
  • any oligonucleotide disclosed herein having one or more 5-methylcytosine LNAs may have any one or more of the 5-methylcytosine LNAs replaced with a cytosine LNA.
  • FIG. 1A is a diagram showing alternative splicing of the FAS gene in the presence of FAS-AS 1 and RBM5.
  • FIG. IB is a diagram showing FAS signaling between naive T cells and memory T cells and FAS-mediated precocious differentiation of naive T cells.
  • FIG. 1C is a diagram showing blocking of FAS signaling upon production of soluble Fas (sFas). See also genevisible.com/tissues/HS/Gene%20Symbol/FAS.
  • FIG. 2 is a graph showing expression of RMB5 in naive T cells and other cell types.
  • the adoptive transfer of specific T cell populations can increase the probability of cancer cell regression and remission (see, e.g., Cieri, N. et al. (2013) IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood 121, 573-584;
  • Naive T (TN) cells are the precursors of T stem cell memory cells (TSCM), T effector memory cells (TEMX and T central memory cells (TCM)-
  • TSCM and TCM populations may increase antitumor, antibacterial, and antiviral responses following adoptive cell transfer in preclinical models.
  • Interaction between memory T cells (T Me m) and T N cells may contribute to proliferation and expansion of the T N cells (see, e.g., Klebanoff, C. A. et al. (2016) Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest 126, 318-334).
  • TMem cells may induce precocious differentiation of the T N cells, thereby limiting the number of the desired T SCM and T CM populations that can be produced from the depleted T N cell population.
  • This precocious differentiation is mediated through FasL (CD95L) and Fas (CD95) interaction on the TMem and T cells, respectively.
  • Blocking of this interaction through a recombinant leucine zipper- dimerized FasL results in an increase of T N cells with proliferative capacity and to increase tumor regression upon adoptive transfer (see, e.g., Klebanoff, C. A. et al. (2016) Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest 126, 318-334).
  • the FAS gene encodes both a membrane-bound form and a soluble form of Fas (mFas and sFas, respectively).
  • the form of Fas protein that is produced in a cell is mediated by alternative splicing of exon 6 of the FAS mRNA. Inclusion of exon 6 results in the production of mFas, whereas exclusion of exon 6 results in the production of sFAS (FIG. 1A).
  • FALS FasL to mFas mediates the differentiation of T N cells described above (FIG. IB and 1C).
  • FAS-AS 1 a long noncoding RNA (IncRNA) that is antisense to the FAS gene, binds to RBM5 (RNA-binding protein 5), which is involved in alternative splicing of exon 6 of FAS.
  • RBM5 RNA-binding protein 5
  • FAS-AS 1 binding inhibits RBM5 activity and results in the inclusion of exon 6 and the production of mFAS (see, e.g., Sehgal, L. et al. (2014) FAS-antisense 1 IncRNA and production of soluble versus membrane Fas in B-cell lymphoma. Leukemia 28, 2376-2387).
  • compositions and methods for maintaining or increasing the number of T N cells in a T cell population by inhibiting the interaction of FAS-ASl with RBM5.
  • the interaction is inhibited using an oligonucleotide as described herein.
  • inhibiting the interaction of FAS-AS l with RBM5 in T N cells will lead to the production of increased levels of sFAS and/or decreased levels of mFAS based on the change in exon 6 splicing.
  • Both RBM5 and FAS-AS l are expressed in T cells (FIGs. 2 and 3). Consequently, in some embodiments, less mFAS is produced by the T N cells, which may result in less FAS-mediated signaling and less precocious differentiation of the T cells.
  • increased levels of sFAS may act as an inhibitor of FasL signaling from the T Mem population, again resulting in less precocious differentiation of the T N cells.
  • inhibiting the interaction of FAS-AS 1 with RBM5 will cause a greater number of T N cells to be present in a T cell population, which, if used in adoptive T cell transfer for cancer treatment, may result in a more effective antitumor response.
  • treatment of a T cell population ex vivo with an oligonucleotide as described herein may result in effects that are limited in time, as the oligonucleotide may be diluted out as the T cell population expands, e.g., once the T cell population is administered to a subject.
  • this temporary window effectiveness may be advantageous, as mFas/FasL signaling is important for in vivo T cell differentiation into effector cells and apoptosis of T cells, which may result in greater efficacy of adoptive T cell transfer in vivo as well as prevent unwanted negative effects caused by T cells that are resistant to apoptosis.
  • the disclosure provides methods for maintaining or increasing the number of naive T cells and/or increasing soluble Fas cell surface death receptor (sFas) in a T cell population.
  • the method comprises administering to a T cell population an oligonucleotide as described herein, e.g., that inhibits the interaction of FAS- AS 1 with RBM5.
  • the administration of the oligonucleotide is ex vivo.
  • the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of
  • the concentration of oligonucleotide delivered to the T cell population is up to 1 ⁇ , up to 5 ⁇ , up to 10 ⁇ , up to 20 ⁇ , up to 50 ⁇ , or up to 100 ⁇ . It is understood that any reference to uses of compounds (e.g., oligonucleotides, expression vectors, inhibitors) throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition or a disease (e.g., cancer) where maintaining or increasing the number of naive T cells in a T cell population is therapeutically beneficial, e.g., in adoptive T cell transfer.
  • a disease e.g., cancer
  • delivering an oligonucleotide as described herein results in an increase in sFas expression in the T cell population (e.g., in the naive T cells) compared to a control level of sFas expression, such as sFas expression in a control T cell population (e.g., in control naive T cells) to which the oligonucleotide has not been delivered.
  • a level of sFas expression may be determined using any suitable assay known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001;
  • the sFas expression level may be an mRNA level or a protein level.
  • sequences of FAS mRNAs and proteins known in the art see, e.g., NCBI Transcript IDs: NM_000043.5, NM_001320619.1,
  • NM_152871.3, and NM_152872.3, and NCBI Protein IDs: NP_000034.1, NP_001307548.1, NP_690610.1, and NP_690611.1) may used to design suitable reagents and assays for measuring an sFas expression level.
  • an appropriate control level of sFas expression may be, e.g., a level of sFas expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered (e.g., a scrambled oligo, a carrier, etc.).
  • an appropriate control level of sFas expression may be a predetermined level or value, such that a control level need not be measured every time.
  • the predetermined level or value can take a variety of forms. It can be single cut-off value, such as a median or mean.
  • increasing sFas expression in a cell includes a level of sFas expression that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above an appropriate control level of sFas.
  • the appropriate control level may be a level of sFas expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein.
  • administration of an oligonucleotide as described herein results in a decreased level of FAS -AS 1 or steric interference with the interaction of FAS- AS 1 with RBM5.
  • decreasing a level of FAS-AS 1 includes a level of FAS- AS 1 that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than an appropriate control level of FAS-AS 1.
  • the appropriate control level may be a level of FAS-AS 1 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein.
  • a T cell population is obtained and an oligonucleotide as described herein is delivered ex vivo to the T cell population.
  • the T cell population is obtained by isolating T cells from a sample (e.g., a blood sample) obtained from a donor subject (e.g., a human donor subject).
  • the T cell population is further enriched, e.g., by selecting T cells expressing certain cell surface markers, such as CD4 and/or CD8.
  • the T cell population is enriched for naive T cells, e.g., by selection for CD4-positive T cells. The selection of T cells may be accomplished using any method known in the art or described herein, e.g., by fluorescence activated cell sorting or magnetic cell sorting.
  • a T cell population to which an oligonucleotide as described herein has been delivered is administered to a host subject (e.g., a human host subject). In some embodiments, this process is referred to as adoptive T cell transfer.
  • the T cell population administered to the subject may further be engineered, prior to administration to the subject, to express a recombinant receptor such as a chimeric antigen receptor (CAR) as described herein.
  • a recombinant receptor such as a chimeric antigen receptor (CAR) as described herein. Suitable administration routes for delivery of the T cell population to a subject are described herein.
  • oligonucleotides are provided for maintaining or increasing the number of naive T cells in a T cell population, e.g., by inhibiting the interaction of FAS-AS 1 with RBM5.
  • expression of soluble Fas (sFas) is upregulated or increased.
  • the oligonucleotide comprises a region of complementarity that is complementary with FAS- AS 1.
  • An exemplary sequence of FAS-AS 1 sequence is provided below:
  • Single stranded oligonucleotides may include secondary structures, e.g., a loop or helix structure.
  • the oligonucleotide comprises at least one modified nucleotide or modified internucleoside linkage as described herein.
  • the oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine
  • oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
  • the oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene.
  • a threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
  • the oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content.
  • the oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content.
  • all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of FAS-AS 1 are cytosine or guanosine nucleotides.
  • the sequence of the FAS-AS l to which the oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
  • the region of complementarity of the oligonucleotide is complementary with 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of FAS-AS l.
  • the region of complementarity is complementary with at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides of FAS-AS l, optionally wherein the oligonucleotide is 8 to 30 nucleotides in length.
  • Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an
  • oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of FAS-AS l, then the oligonucleotide and FAS-AS l are considered to be complementary to each other at that position.
  • the oligonucleotide and FAS-AS l are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases.
  • complementary is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and FAS-ASl . For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of FAS-AS l, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • the oligonucleotide may be at least 70% complementary to (optionally one of at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of FAS-AS 1.
  • the oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of FAS -AS 1.
  • the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
  • a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target molecule.
  • a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable or specific for the target molecule when binding of the sequence to the target molecule (e.g., FAS-AS 1) interferes with the normal function of the target (e.g., FAS-AS 1) to cause a loss of activity (e.g., inhibiting the interaction with RBM5) or expression (e.g., degrading the FAS-AS 1) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays, ex vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • the oligonucleotide is up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 35, up to 40, up to 45, or up to 50 nucleotides in length.
  • the oligonucleotide is 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, 10 to 50, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15 nucleotides in length.
  • the oligonucleotide is 8 to 30 nucleotides in length.
  • Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa.
  • GC content of the oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
  • the oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof.
  • the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; or have improved endosomal exit.
  • oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker.
  • a linker e.g., a cleavable linker.
  • Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention may include a phosphorothioate at least the first, second, or third internucleoside linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2 -O-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0- DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0--N-methylacetamido (2'-0— NMA).
  • a 2'-modified nucleotide e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2 -O-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl
  • the nucleic acid sequence can include at least one 2'-0- methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0- methyl modification.
  • the nucleic acids are "locked,” i.e., comprise nucleic acid analogues in which the ribose ring is "locked” by a methylene bridge connecting the 2'-0 atom and the 4'-C atom.
  • any of the modified chemistries or formats of oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
  • an oligonucleotide may comprise one or more modified nucleotides (also referred to herein as nucleotide analogs).
  • the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide.
  • the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide.
  • LNA locked nucleic acid
  • cEt constrained ethyl
  • ENA ethylene bridged nucleic acid
  • the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes.
  • the oligonucleotide may have one or more 2' O-methyl nucleotides.
  • the oligonucleotide may consist entirely of 2' O-methyl nucleotides.
  • the oligonucleotide has one or more nucleotide analogues.
  • the oligonucleotide may have at least one nucleotide analogue that results in an increase in T m of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an
  • the oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in T m of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
  • the oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
  • the oligonucleotide may consist entirely of bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides).
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides.
  • the oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides.
  • the oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide).
  • the oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
  • the oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the oligonucleotide may comprise
  • deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the 3' position of the oligonucleotide may have a 3' hydroxyl group.
  • the 3' position of the oligonucleotide may have a 3' thiophosphate.
  • the oligonucleotide may be conjugated with a label.
  • the oligonucleotide may be conjugated with a label.
  • oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • a biotin moiety cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • the oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
  • the oligonucleotides are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • beneficial properties such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target
  • Chimeric oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • the oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-0-alkyl, 2'-0-alkyl-0-alkyl or 2'-fluoro- modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as
  • oligonucleotides may have phosphorothioate backbones; heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see
  • PNA peptide nucleic acid
  • Phosphorus -containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • the oligonucleotide is an oligonucleotide mimetic.
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001;
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos.
  • Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
  • Arabinonucleosides are stereoisomers of ribonucleo sides, differing only in the configuration at the 2'-position of the sugar ring.
  • a 2'-arabino modification is 2'-F arabino.
  • the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
  • ENAs ethylene -bridged nucleic acids
  • Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene-bridged nucleic acids. Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.
  • -CH CH-, where R is selected from hydrogen and Ci_ 4 -alkyl; Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
  • the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
  • Y is -0-, -S-, -NH-, or N(R ); Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and Ci_ 4 -alkyl.
  • the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
  • the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O) 2 -O-, -0-P(0,S)-0-, -0-P(S) 2 -O-, -S-P(0) 2 -0-, -S-P(0,S)-0-, -S-P(S) 2 -0-, -0-P(0) 2 -S-, -0-P(0,S)-S-, -S-P(0) 2 -S-, -0-PO(R H )-0-, o- PO(OCH 3 )-0-, -0-PO(NR H )-0-, -0-PO(OCH 2 CH 2 S-R)-O-, -0-PO(BH 3 )-0-, -0-PO(NHR H )- 0-, -0-P(0) 2 -NR H -, -NR H -P(0) 2 -0-, -NR H -CO-0-0-
  • LNA units are shown below:
  • thio-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH 2 -S-.
  • Thio-LNA can be in both beta-D and alpha-L-configuration.
  • amino-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 -N(R)- where R is selected from hydrogen and Ci_ 4 -alkyl.
  • Amino-LNA can be in both beta-D and alpha-L-configuration.
  • oxygen-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH 2 -0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
  • ena-LNA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
  • LNAs are described in additional detail herein.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a
  • a preferred modification includes 2'-methoxyethoxy [2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Other preferred modifications include 2'- methoxy (2'-0-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3 ) and 2'-fluoro (2'-F). Similar
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine,
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with modified groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • Oligonucleotides can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • base any nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • “unmodified” or “natural” nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo -uracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-sub
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • the oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • one or more oligonucleotides, of the same or different types can be conjugated to each other; or oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg.
  • a thioether e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
  • an aliphatic chain e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate
  • a phospholipid e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether,
  • oligonucleotide modification includes modification of the 5' or 3' end of the oligonucleotide.
  • the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate.
  • additional molecules e.g. a biotin moiety or a fluorophor
  • the oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
  • the oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'- fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'-0-methyl nucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides.
  • LNA locked nucleic acids
  • ENA modified nucleotides 2'-0-methyl nucleotides
  • 2'-fluoro-deoxyribonucleotides In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'- fluor
  • the oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
  • the 5' nucleotide of the oligonucleotide is a
  • the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide.
  • the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3 ' ends of the deoxyribonucleotides.
  • the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.
  • the oligonucleotide comprises phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises
  • the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides.
  • oligonucleotide can have any combination of modifications as described herein.
  • an oligonucleotide described herein may be a mixmer or comprise a mixmer sequence pattern.
  • the term 'mixmer' refers to oligonucleotides which comprise both naturally and non-naturally occurring nucleotides or comprise two different types of non-naturally occurring nucleotides.
  • Mixmers are generally known in the art to have a higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule. Generally, mixmers do not recruit an RNAse to the target molecule and thus do not promote cleavage of the target molecule.
  • the mixmer comprises or consists of a repeating pattern of nucleotide analogues and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogue.
  • the mixmer need not comprise a repeating pattern and may instead comprise any arrangement of nucleotide analogues and naturally occurring nucleotides or any arrangement of one type of nucleotide analogue and a second type of nucleotide analogue.
  • a pattern in general, refers to a pattern of modifications or lack thereof, and not to a pattern of A, T, C, G, or U nucleotides.
  • the repeating pattern may, for instance be every second or every third nucleotide is a nucleotide analogue, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2' substituted nucleotide analogue such as 2'-0-methyl, 2'MOE or 2' fluoro analogues, or any other nucleotide analogues described herein. It is recognized that the repeating pattern of nucleotide analogues, such as LNA units, or 2'-0-methyl, 2'MOE or 2' fluoro analogues, may be combined with nucleotide analogues at fixed positions— e.g. at the 5' or 3 ' termini.
  • the mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleotides, such as DNA nucleotides.
  • the mixmer comprises at least a region consisting of at least two consecutive nucleotide analogues, such as at least two consecutive LNAs.
  • the mixmer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNAs.
  • the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogues, such as LNAs. It is to be understood that the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
  • the mixmer comprises at least one nucleotide analogue in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occurring nucleotide, such as DNA or RNA.
  • the mixmer comprises at least two nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, xXXxxx, xXxXxx, xXxxxX, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxXx, xxxXxX and xxxxXX, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x” denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxxxX, xxXxXx, xxXxxX and xxxXxX.
  • the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX.
  • the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx.
  • the substitution pattern for the nucleotides is xXxXxx.
  • the mixmer comprises at least three nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXX, XxxxXX, XxxxXX, xXxXXx, xXxxXXX, xxXXX, xXxXxX and XxXxXx, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x” denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the substitution pattern for the nucleotides is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxxxXX, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx.
  • the substitution pattern for the nucleotides is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX. n some embodiments, the substitution pattern for the nucleotides is xXxXxX or XxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxX.
  • the mixmer comprises at least four nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of xXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXx, XxxXXX, XxXxX, XxXXxX, XxXXx, XXxxXX, XXxXxX, XXxXx, XXxxX, XXXxXx and XXXXxx, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the mixmer comprises at least five nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX,
  • XXXXxX and XXXXx wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
  • the mixmer contains a modified nucleotide, e.g., an LNA, at the 5' end. In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the first two positions, counting from the 5' end.
  • the mixmer is incapable of recruiting RNAseH.
  • Oligonucleotides that are incapable of recruiting RNAseH are well known in the literature, in example see WO2007/112754, WO2007/112753, or PCT/DK2008/000344.
  • Mixmers may be designed to comprise a mixture of affinity enhancing nucleotide analogues, such as in non- limiting example LNA nucleotides and 2'-0-methyl nucleotides.
  • the mixmer comprises modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • a mixmer is 4 to 40 nucleotides (e.g., 4 to 40, 4 to 35, 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10), in length having the general formula:
  • each instance of X 4 is a modified or unmodified nucleotide described herein (e.g., a modified or unmodified ribonucleotide described herein), wherein each instance of X s is a deoxyribonucleotide, wherein p and q are independently 0 or 1, reflecting the number of instances of X 1 and X 2 , respectively, wherein at least one of X 1 and X 2 is present in each instance of the unit, ⁇ X p — X q wherein r is an integer from 2 to 20 reflecting the number of instances of the unit, ⁇ X p — X ⁇ , linked together through internucleotide linkages, wherein the protecting oligonucleotide or region does not contain a sequence of more than 5 consecutive deoxyribonucleotides, and wherein the symbol "— " denotes an internucleotide linkage.
  • a mixmer may be produced using any method known in the art or described herein.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646,
  • the oligonucleotide is a gapmer.
  • the gapmer has a sequence following the general formula:
  • each instance of X , X 3 is independently a modified or unmodified nucleotide described herein (e.g., a modified or unmodified ribonucleotide described herein), wherein m and o are independently integers in a range of 1 to 10 (e.g., 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 1 to 6, 2 to 6, 3 to 6, or 4 to 6) reflecting the number of instances of X 1 and X 3 , respectively, linked consecutively together through internucleotide linkages, wherein each instance of X 2 is a deoxyribonucleotide, wherein n is an integer in a range of 6 to 20 (e.g., 6 to 20, 6 to 15, 6 to 10, 7 to
  • a gapmer oligonucleotide may also have the formula 5'-X-Y-Z-3', with X and Z as flanking regions around a gap region Y.
  • the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAseH.
  • RNAseH an RNAse
  • the Y region is flanked both 5' and 3' by regions X and Z comprising high-affinity modified nucleotides, e.g., 1 - 6 modified nucleotides.
  • exemplary modified oligonucleotides include, but are not limited to, 2' MOE or 2'OMe or Locked Nucleic Acid bases (LNA).
  • the flanks X and Z may be have a of length 1 - 20 nucleotides, preferably 1-8 nucleotides and even more preferred 1 - 5 nucleotides.
  • the flanks X and Z may be of similar length or of dissimilar lengths.
  • the gap-segment Y may be a nucleotide sequence of length 5 - 20 nucleotides, preferably 6-12 nucleotides and even more preferred 6 - 10 nucleotides.
  • the gap region of the gapmer oligonucleotides of the invention may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides.
  • the gap region comprises one or more unmodified internucleosides.
  • flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • a gapmer may be produced using any method known in the art or described herein.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. patent publication Nos. US20090286969, US20100197762, and US20110112170; and PCT publication Nos.
  • oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA.
  • siRNA is a class of double- stranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective.
  • siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence can be designed and prepared using any method known in the art (see, e.g., PCT Publication Nos. WO08124927A1 and WO 2004/016735; and U.S. Patent
  • a number of commercial packages and services are available that are suitable for use for the preparation of siRNA molecules. These include the in vitro transcription kits available from Ambion (Austin, TX) and New England Biolabs (Beverly, MA) as described above; viral siRNA construction kits commercially available from Invitrogen (Carlsbad, CA) and Ambion (Austin, TX), and custom siRNA construction services provided by Ambion (Austin, TX), Qiagen (Valencia, CA), Dharmacon (Lafayette, CO) and Sequitur, Inc (Natick, MA).
  • a target sequence can be selected (and a siRNA sequence designed) using computer software available commercially (e.g.
  • an siRNA may be designed or obtained using the RNAi atlas (available at the RNAiAtlas website), the siRNA database (available at the Swedish Bioinformatics Website), or using DesiRM (available at the Institute of Microbial
  • the siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand).
  • the siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense strands.
  • Double- stranded siRNA may comprise RNA strands that are the same length or different lengths.
  • Double-stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single- stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
  • Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end and/or the 5' end of either or both strands).
  • shRNA Small hairpin RNA
  • a spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double- stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands).
  • a spacer sequence is may be an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a shRNA.
  • the overall length of the siRNA molecules can vary from about 14 to about 200 nucleotides depending on the type of siRNA molecule being designed. Generally between about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single- stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 200 nucleotides.
  • siRNA molecule may comprise a 3' overhang at one end of the molecule, The other end may be blunt-ended or have also an overhang (5' or 3') ⁇
  • the siRNA molecule of the present invention comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.
  • an oligonucleotide may be a microRNA (miRNA).
  • MicroRNAs are small non-coding RNAs, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on a target RNA transcript.
  • miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre- miRNAs, which fold into imperfect stem- loop structures.
  • pri-miRNAs large RNA precursors
  • pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.
  • miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA.
  • the size range of the miRNA can be from 21 nucleotides to 170 nucleotides, although miRNAs of up to 2000 nucleotides can be utilized. In one embodiment the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miPvNAs of from 21 to 25 nucleotides in length can be used.
  • a miRNA is expressed from a vector.
  • the vector may include a sequence encoding a mature miRNA.
  • the vector may include a sequence encoding a pre-miRNA such that the pre-miRNA is expressed and processed in a cell into a mature miRNA.
  • the vector may include a sequence encoding a pri-miRNA.
  • the primary transcript is first processed to produce the stem-loop precursor miRNA molecule. The stem-loop precursor is then processed to produce the mature microRNA.
  • oligonucleotides provided herein may be in the form of aptamers.
  • An "aptamer” is any nucleic acid that binds specifically to a target, such as a small molecule, protein, nucleic acid, cell, tissue or organism.
  • the aptamer is a DNA aptamer or an RNA aptamer.
  • a nucleic acid aptamer is a single- stranded DNA or RNA (ssDNA or ssRNA). It is to be understood that a single- stranded nucleic acid aptamer may form helices and/or loop structures.
  • the nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or a combination of thereof.
  • hydrocarbon linkers e.g., an alkylene
  • a polyether linker e.g., a PEG linker
  • nucleic acid aptamers may be accomplished by any suitable method known in the art, including an optimized protocol for in vitro selection, known as SELEX (Systemic Evolution of Ligands by Exponential enrichment). Many factors are important for successful aptamer selection. For example, the target molecule should be stable and easily reproduced for each round of SELEX, because the SELEX process involves multiple rounds of binding, selection, and amplification to enrich the nucleic acid molecules. In addition, the nucleic acids that exhibit specific binding to the target molecule have to be present in the initial library. Thus, it is advantageous to produce a highly diverse nucleic acid pool. Because the starting library is not guaranteed to contain aptamers to the target molecule, the SELEX process for a single target may need to be repeated with different starting libraries.
  • SELEX Systemic Evolution of Ligands by Exponential enrichment
  • Exemplary publications and patents describing aptamers and method of producing aptamers include, e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT application WO 99/31275, each incorporated herein by reference.
  • oligonucleotides provided herein may be in the form of a ribozyme.
  • a ribozyme ribonucleic acid enzyme
  • Ribozymes are molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, IncRNAs, and ribozymes, themselves.
  • Ribozymes may assume one of several physical structures, one of which is called a "hammerhead.”
  • a hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double- stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions the catalytic core. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double- stranded stems I and III.
  • Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to a specific ribonucleotide triplet by a transesterification reaction from a 3', 5'-phosphate diester to a 2', 3 '-cyclic phosphate diester.
  • this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
  • Ribozyme oligonucleotides can be prepared using well known methods (see, e.g., PCT Publications W09118624; W09413688; WO9201806; and WO 92/07065; and U.S.
  • Patents 5436143 and 5650502 can be purchased from commercial sources (e.g., US Biochemicals) and, if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by nucleases in a cell.
  • the ribozyme may be synthesized in any known manner, e.g., by use of a commercially available synthesizer produced, e.g., by Applied Biosystems, Inc. or Milligen.
  • the ribozyme may also be produced in recombinant vectors by conventional means. See, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition).
  • the ribozyme RNA sequences maybe synthesized conventionally, for example, by using RNA polymerases such as T7 or SP6.
  • the oligonucleotide is an oligonucleotide mimetic, such as a morpholino -based oligomeric compound, a cyclohexenyl nucleic acid oligonucleotide mimetics, or peptide nucleic acid (PNA) compound.
  • oligonucleotide mimetic such as a morpholino -based oligomeric compound, a cyclohexenyl nucleic acid oligonucleotide mimetics, or peptide nucleic acid (PNA) compound.
  • methods are provided for producing candidate
  • oligonucleotides that are useful for, e.g., inhibiting the interaction of FAS-AS1 with RBM5.
  • the oligonucleotides are complementary to sequences in a target RNA, e.g., FAS- AS 1.
  • the oligonucleotides are designed by determining a region of a target RNA (FAS -AS 1); producing an oligonucleotide that has a region of complementarity that is complementary with a plurality of (e.g., at least 5) contiguous nucleotides of the region of the target RNA; and determining whether administering the oligonucleotide to a cell in which FAS-AS 1 and RBM5 are expressed results in inhibition of the interaction and/or increased levels of soluble Fas.
  • FAS -AS 1 target RNA
  • methods are provided for obtaining one or more
  • oligonucleotides for inhibiting the interaction of FAS-AS 1 with RBM5 that further involve producing a plurality of different oligonucleotides, in which each oligonucleotide has a region of complementarity that is complementary with a plurality of (e.g., at least 5) contiguous nucleotides in a target RNA (e.g., FAS-AS1); subjecting each of the different oligonucleotides to an assay that assesses whether delivery of an oligonucleotide to a cell harboring the target gene results in inhibition of the interaction and/or increased levels of soluble Fas in the cell; and obtaining one or more oligonucleotides that inhibit the interaction and/or increase levels of soluble Fas in the assay.
  • a target RNA e.g., FAS-AS1
  • a T cell population described herein e.g., comprising naive T cells
  • an expression construct encoding a CAR is transfected with an expression construct encoding a CAR.
  • CARs have been utilized to engineer T cells to target various antigens, such as tumor antigens.
  • CARs comprise an extracellular antigen-binding domain (e.g., a single chain variable fragment (scFv) from an antibody), a transmembrane domain (e.g., a transmembrane domain of any one of the following: alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, and CD154) and an intracellular domain comprising one or more signaling or co- stimulatory domains (e.g., one or more signaling domains of the ⁇ 3 ⁇ chain, 4-1BB (CD137) and CD28 and/or one or more co-stimulatory domains of 4-1BB, CD28, ICOS, DAP
  • CARs may further comprise a hinge region such as a IgGl, IgG4, and IgD or CD8 hinge.
  • a hinge region such as a IgGl, IgG4, and IgD or CD8 hinge.
  • Exemplary CARs and methods of making such CARs are known in the art (see, e.g., PCT publication numbers WO2014184744A1,
  • the CAR comprises (a) a scFv specific for an antigen (e.g., a tumor antigen), (b) a hinge region (e.g., an Ig hinge region), (c) a transmembrane domain (e.g., a CD3 ⁇ chain, CD4, CD8, ICOS, or CD28 transmembrane domain), (d) a CD3 ⁇ chain signaling domain and optionally (e) one or more co- stimulatory domains selected from ICOS, OX40 (CD134), CD28, 4-1BB (CD137), CD27, and DAP10.
  • an antigen e.g., a tumor antigen
  • a hinge region e.g., an Ig hinge region
  • a transmembrane domain e.g., a CD3 ⁇ chain, CD4, CD8, ICOS, or CD28 transmembrane domain
  • CD3 ⁇ chain signaling domain e.g., OX40 (CD134), CD28, 4-1BB (CD137),
  • the CAR is specific for a tumor antigen (e.g., contains a scFv specific for a tumor antigen).
  • a tumor antigen e.g., contains a scFv specific for a tumor antigen.
  • exemplary tumor antigens include CD 19, CD20, CD33, HER2, GD2 ganglioside, CD171, CAIX, a-folate receptor, IL13Ra2 and CEA.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross- species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A,” the presence of a molecule containing epitope "A” (or free, unlabeled "A”), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • transfection of the T cell with the CAR expression construct occurs before the T cell population is contacted with the oligonucleotide. In some embodiments, transfection occurs after the T cell population is contacted with the
  • transfection occurs at the same time that the T cell population is contacted with the oligonucleotide.
  • the T cell population is activated prior to transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody optionally immobilized on a solid substrate.
  • the T cell is activated after transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody.
  • transfection is achieved by lentiviral infection of the T cell population with the expression construct encoding the CAR.
  • the expression construct may comprise the coding sequence of the CAR optionally along with one or more regulatory sequences that drive expression of the coding sequence, e.g., a promoter and/or enhancer sequence.
  • the expression construct is a lentiviral construct comprising 5' and 3' long terminal repeats (LTRs).
  • Lentiviruses for use in transfecting T cell populations can be produced using any method known in the art or described herein.
  • 293FT cells may be co- transfected with lentiviral helper plasmids and a lentiviral construct comprising the coding sequence of the CAR optionally with regulatory sequences.
  • Virus supernatants can be isolated from the 293T cells and then concentrated, e.g., by ultracentrifugation.
  • the T cells for use in developing a CAR T cells may be obtained using any method known in the art or described herein (see, e.g., PCT publication numbers WO2014184744A1, WO2014184143A1, WO2014059173A2 and WO2015179801A1).
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells
  • PBMCs bone marrow, lymph node tissue, cord blood, thymus tissue and spleen tissue from a donor subject (e.g., a human donor subject).
  • PBMCs can be obtained, e.g., by FicollTM separation from blood obtained from the donor subject.
  • the T cells may be obtained from a T cell line.
  • a specific subpopulation of T cells, such as CD4 + T cells, can be further isolated by positive or negative selection techniques, such as by fluorescent activated cell sorting or magnetic cell sorting.
  • a T cell population transfected with a CAR expression construct is administered to a host subject (e.g., a human host subject).
  • a host subject e.g., a human host subject.
  • the subject has cancer and the CAR is specific for a tumor antigen expressed by the cancer in the subject.
  • Vectors include, but are not limited to, plasmids, viral vectors, other vehicles derived from viral or bacterial or other sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences for expressing an RNA transcript (e.g., mRNA).
  • expression vectors are provided that are engineered to express a chimeric antigen receptor (CAR) as described herein.
  • an expression vector may be engineered by incorporating a coding sequence for a gene of interest (e.g., a CAR) into a plasmid that is suitably configured with expression elements (e.g., a promoter) for expressing the gene of interest.
  • cDNA may be obtained or synthesized using a commercially available kit or any method known in the art.
  • a vector may comprise one or more expression elements.
  • “Expression elements” are any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient expression of an RNA transcript (e.g., mRNA).
  • the expression element may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter or a tissue specific promoter, examples of which are well known to one of ordinary skill in the art.
  • Constitutive mammalian promoters include polymerase promoters as well as the promoters for the following non- limiting genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, and beta-actin.
  • HPTR hypoxanthine phosphoribosyl transferase
  • adenosine deaminase pyruvate kinase
  • beta-actin beta-actin
  • Exemplary viral promoters which function constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • Other constitutive promoters may be used.
  • Inducible promoters are expressed in the presence of an inducing agent and include metal-inducible promoters and steroid-regulated promoters, for example. Other inducible promoters may be used.
  • Expression vectors may also comprise an origin of replication, a suitable promoter polyadenylation site, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • Viral vectors are generally based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the nucleic acid sequence of interest. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lines with plasmid, production of recombinant retroviruses by the packaging cell lie, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) may be used.
  • Viral and retroviral vectors that may be used include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as any retrovirus.
  • retroviruses such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses
  • compositions e.g., T cell populations that have been contacted with an oligonucleotide
  • routes e.g., T cell populations that have been contacted with an oligonucleotide
  • Exemplary routes include: intrathecal, intraneural, intracerebral, intramuscular, oral, intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, or ocular.
  • therapeutically effective amount is the amount of active agent (e.g., oligonucleotide or T cell population) present in the composition that is needed to provide the desired level of sFas expression in the T cell population or to provide a treatment effect in the subject to be treated, e.g., treatment of cancer.
  • physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect.
  • pharmaceutically acceptable carrier means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically include one or more species of oligonucleotide or T cell population and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • 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 The route and site of administration may be chosen to enhance targeting. For example, to target a tumor, intratumoral injection may be desirable.
  • Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject.
  • the most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
  • the most common topical delivery is to the skin.
  • the term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum.
  • Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition.
  • Topical administration can also be used as a means to selectively deliver compositions to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
  • 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.
  • Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics.
  • the dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin.
  • Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
  • transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.
  • iontophoresis transfer of ionic solutes through biological membranes under the influence of an electric field
  • phonophoresis or sonophoresis use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea
  • optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
  • oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
  • GI gastrointestinal
  • compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek.
  • the sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
  • a pharmaceutical composition may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant.
  • the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
  • compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration.
  • parental administration involves administration directly to the site of disease (e.g., tumor).
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • the total concentration of solutes should be controlled to render the preparation isotonic.
  • Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
  • Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers.
  • the delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • the types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
  • HSA human serum albumin
  • bulking agents such as carbohydrates, amino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
  • Pulmonary administration of a micellar oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non- CFC and CFC propellants.
  • Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
  • Other devices include non-vascular devices, e.g., devices implanted in the
  • the device can release a therapeutic substance in addition to an oligonucleotide.
  • unit doses or measured doses of a composition are dispensed by an implanted device.
  • the device can include a sensor that monitors a parameter within a subject.
  • the device can include pump, e.g., and, optionally, associated electronics.
  • T cell populations described herein can be treated with an oligonucleotide ex vivo and then administered or implanted in a subject.
  • the T cell population can be autologous, allogeneic, or xenogeneic to the subject.
  • Introduction of treated T cell populations, whether autologous or transplant, can be combined with other therapies.
  • the invention features a method of administering an oligonucleotide to a T cell population (e.g., a human T cell population comprising naive T cells).
  • a T cell population e.g., a human T cell population comprising naive T cells.
  • 1 to 40 micromolar (e.g., 1 to 20 micromolar) of oligonucleotide is
  • the invention features a method of administering a T cell population (e.g., a human T cell population comprising naive T cells that has been contacted with an oligonucleotide herein) to a subject (e.g., a human subject).
  • a T cell population e.g., a human T cell population comprising naive T cells that has been contacted with an oligonucleotide herein
  • the dosage in between 10 4 to 10 9 cells/kg body weight.
  • T cell populations may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • the defined amount can be an amount effective to upregulate sFas expression in naive T cells in a T cell population.
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder that would benefit from adoptive T cell transfer, such as cancer.
  • the unit dose is administered or delivered daily. In some embodiments, less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered or delivered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered or delivered a single time. In some embodiments, the unit dose is administered or delivered more than once a day, e.g., once an hour, two hours, four hours, eight hours, twelve hours, etc. Further, the treatment regimen for T cell populations 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.
  • the subject can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage may either be increased in the event the subject 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, if the disease state has been ablated, or if undesired side-effects are observed.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semipermanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • a delivery device e.g., a pump, semipermanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage may increase or decrease over the course of a particular treatment.
  • Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models.
  • the administration of a composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ocular, intraneuronal, intrathecal, or intracerebral.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • kits comprising a container housing a composition comprising an oligonucleotide as described herein.
  • the composition is a pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier.
  • the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for oligonucleotides, and at least another for a carrier compound.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g., according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • Gapmer oligonucleotides were designed to target FAS-AS 1.
  • the sequence and chemistry of each gapmer oligonucleotide is shown in Table 1.
  • Table 2 provides a description of the nucleotide analogs, modifications and internucleoside linkages used for certain oligonucleotides described in Table 1.
  • GTGTAAGAAAATTGT 8 lnaGs;lnaTs;lnaGs;dTs;dAs;dAs;dGs;d
  • TTTATTGTCACACAG 26 lnaTs;lnaTs;lnaTs;dAs;dTs;dTs;dGs;dT s;dCs;dAs;dCs;dAs;lnaCs;lnaAs;lnaG
  • CTGCCTTGTCTCCCT 28 lnaCs;lnaTs;lnaGs;dCs;dCs;dTs;dTs;dG s;dTs;dCs;dTs;dCs;lnaCs;lnaCs;lnaT
  • TCCGGGAATTCTCTC 29 lnaTs;lnaCs;lnaCs;dGs;dGs;dGs;dAs;d
  • CTCGTTCCCACCGCA 42 lnaCs;lnaTs;lnaCs;dGs;dTs;dTs;dCs;dC s;dCs;dAs;dCs;dCs;lnaGs;lnaCs;lnaA
  • GCGCCTATTATTGGC 44 lnaGs;lnaCs;lnaGs;dCs;dCs;dTs;dAs;dT s;dTs;dAs;dTs;dTs;lnaGs;lnaGs;lnaC
  • TTTGAGTACCGGAGC 53 lnaTs;lnaTs;lnaTs;dGs;dAs;dGs;dTs;dA s;dCs;dCs;dGs;dGs;lnaAs;lnaGs;lnaC
  • AATTCCAAAACTCAG 72 lnaAs;lnaAs;lnaTs;dTs;dCs;dCs;dAs;dA s;dAs;dAs;dCs;dTs;lnaCs;lnaAs;lnaG
  • CCTTTCAGAAATAGT 80 lnaCs;lnaCs;lnaTs;dTs;dTs;dCs;dAs;dG s;dAs;dAs;dAs;dTs;lnaAs;lnaGs;lnaT
  • AATGATTCAAGATTG 85 lnaAs;lnaAs;lnaTs;dGs;dAs;dTs;dTs;dC s;dAs;dAs;dGs;dAs;lnaTs;lnaTs;lnaG
  • TTATACAACCTCAGG 87 lnaTs;lnaTs;lnaAs;dTs;dAs;dCs;dAs;dA s;dCs;dTs;dCs;lnaAs;lnaGs;lnaG
  • GCCACACTCTTCTCT 88 lnaGs;lnaCs;lnaCs;dAs;dCs;dAs;dCs;dT s;dCs;dTs;dCs;lnaTs;lnaCs;lnaT
  • GCTTTGAAAATCTCA 92 lnaGs;lnaCs;lnaTs;dTs;dTs;dGs;dAs;dA s;dAs;dAs;dTs;dCs;lnaTs;lnaCs;lnaA
  • GAACTTTTGTACCAA 98 lnaGs;lnaAs;lnaAs;dCs;dTs;dTs;dT s;dGs;dTs;dAs;dCs;lnaCs;lnaAs;lnaAs;lnaA
  • ACACACACGCATATG 104 lnaAs;lnaCs;lnaAs;dCs;dAs;dCs;dAs;d
  • GTAAATATTCATACA 105 lnaGs;lnaTs;lnaAs;dAs;dAs;dTs;dAs;dT s;dTs;dCs;dAs;dTs;lnaAs;lnaCs;lnaA CATTTATGTATATAT 106 lnaCs;lnaAs;lnaTs;dTs;dTs;dAs;dTs;dG s;dTs;dAs;dTs;dAs;lnaTs;lnaAs;lnaT
  • AACTTATATTTGTAT 109 lnaAs;lnaAs;lnaCs;dTs;dTs;dAs;dTs;dA s;dTs;dTs;dTs;dGs;lnaTs;lnaAs;lnaT
  • ATGATATATGGCCTA 110 lnaAs;lnaTs;lnaGs;dAs;dTs;dAs;dTs;dA s;dTs;dGs;dGs;dCs;lnaCs;lnaTs;lnaA
  • CTAGGAAATTAAGGC 111 lnaCs;lnaTs;lnaAs;dGs;dGs;dAs;dAs;d
  • CATAACTCTATCACC 116 lnaCs;lnaAs;lnaTs;dAs;dAs;dCs;dTs;dC s;dTs;dAs;dTs;dCs;lnaAs;lnaCs;lnaC
  • TCACCTAAGTAATCA 117 lnaTs;lnaCs;lnaAs;dCs;dCs;dTs;dAs;dA s;dGs;dTs;dAs;dAs;lnaTs;lnaCs;lnaA
  • TGCAGTTTATCTTCC 119 lnaTs;lnaGs;lnaCs;dAs;dGs;dTs;dTs;dT s;dAs;dTs;dCs;dTs;lnaTs;lnaCs;lnaC
  • CCATTTCTCCCCTCT 120 lnaCs;lnaCs;lnaAs;dTs;dTs;dTs;dCs;dT s;dCs;dCs;dCs;dCs;lnaTs;lnaCs;lnaT
  • CTTC ACG GTTATGTT 132 lnaCs;lnaTs;lnaTs;dCs;dAs;dCs;dGs;dG
  • oligonucleotides in Table 3 each have the structural motif of
  • InaNs are LNA nucleotides and dNs are DNA nucleotides and the nucleotides are all linked by phosphorothioate linkages as indicated by the "s" after each nucleotide.
  • InaCs are 5- methylcytosine LNAs. Table 3: Further gapmer oligonucleotides

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des compositions et des procédés pour maintenir ou augmenter le nombre de lymphocytes T naïfs dans une population de lymphocytes T, par exemple, par l'administration ex vivo d'un oligonucléotide qui inhibe l'interaction de FAS-AS I avec RBM5 à une population de lymphocytes T comprenant des lymphocytes T naïfs. De telles compositions et procédés sont utiles, par exemple, dans des thérapies adoptives à lymphocytes T, par exemple des thérapies à lymphocytes T portant un récepteur antigénique chimérique (CAR).
PCT/US2018/047473 2017-08-22 2018-08-22 Modulation de l'expression de fas soluble WO2019040590A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762548483P 2017-08-22 2017-08-22
US62/548,483 2017-08-22

Publications (1)

Publication Number Publication Date
WO2019040590A1 true WO2019040590A1 (fr) 2019-02-28

Family

ID=65439873

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/047473 WO2019040590A1 (fr) 2017-08-22 2018-08-22 Modulation de l'expression de fas soluble

Country Status (1)

Country Link
WO (1) WO2019040590A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023031394A1 (fr) 2021-09-03 2023-03-09 CureVac SE Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2023073228A1 (fr) 2021-10-29 2023-05-04 CureVac SE Arn circulaire amélioré pour exprimer des protéines thérapeutiques
WO2023144330A1 (fr) 2022-01-28 2023-08-03 CureVac SE Inhibiteurs de facteurs de transcription codés par un acide nucleique
WO2023227608A1 (fr) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Vaccin à base d'acide nucléique codant pour un polypeptide antigénique fimh d'escherichia coli
DE202023106198U1 (de) 2022-10-28 2024-03-21 CureVac SE Impfstoff auf Nukleinsäurebasis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014039044A1 (fr) * 2012-09-06 2014-03-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de production de populations de cellules souches t mémoires

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014039044A1 (fr) * 2012-09-06 2014-03-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de production de populations de cellules souches t mémoires

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AUNE, THOMAS M. ET AL.: "Long noncoding RNAs in T lymphocytes", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 99, January 2016 (2016-01-01), pages 31 - 44, XP055580547 *
DESBARATS, J. ET AL.: "Dichotomy between naive and memory CD 4+ T cell responses to Fas engagement", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 96, July 1999 (1999-07-01), pages 8104 - 8109, XP055580499 *
KLEBANOFF, CHRISTOPHER A. ET AL.: "Memory T cell -driven differentiation of naive cells impairs adoptive immunotherapy", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 126, no. 1, January 2016 (2016-01-01), pages 318 - 334, XP055580492 *
SEHGAL, L. ET AL.: "FAS-antisense 1 IncRNA and production of soluble versus membrane Fas in B- cell lymphoma", LEUKEMIA, vol. 28, 2014, pages 2376 - 2387, XP055580494 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023031394A1 (fr) 2021-09-03 2023-03-09 CureVac SE Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2023073228A1 (fr) 2021-10-29 2023-05-04 CureVac SE Arn circulaire amélioré pour exprimer des protéines thérapeutiques
WO2023144330A1 (fr) 2022-01-28 2023-08-03 CureVac SE Inhibiteurs de facteurs de transcription codés par un acide nucleique
WO2023227608A1 (fr) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Vaccin à base d'acide nucléique codant pour un polypeptide antigénique fimh d'escherichia coli
DE202023106198U1 (de) 2022-10-28 2024-03-21 CureVac SE Impfstoff auf Nukleinsäurebasis

Similar Documents

Publication Publication Date Title
AU2018202634B2 (en) Multimeric oligonucleotide compounds
AU2019200789B2 (en) Multimeric oligonucleotide compounds
US10041074B2 (en) Euchromatic region targeting methods for modulating gene expression
US20150225722A1 (en) Methods for selective targeting of heterochromatin forming non-coding rna
WO2019040590A1 (fr) Modulation de l'expression de fas soluble
EP3033423A1 (fr) Régulateurs épigénétiques de la frataxine
EP3033425A1 (fr) Compositions et procédés de modulation de l'expression de la frataxine
KR20160074368A (ko) Utrn 발현을 조절하기 위한 조성물 및 방법
JP2016521556A (ja) Ｆｏｘｐ３発現を調節するための組成物及び方法
WO2016130943A1 (fr) Oligonucléotides hybrides et leurs utilisations
EP3256590A1 (fr) Oligonucléotides de ciblage et utilisations de ceux-ci pour moduler l'expression génique
WO2018031871A1 (fr) Modulation ex vivo de l'expression de foxp3

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18847999

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18847999

Country of ref document: EP

Kind code of ref document: A1