WO2014090985A1 - Modulation des lymphocytes t par saut d'exon - Google Patents

Modulation des lymphocytes t par saut d'exon Download PDF

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Publication number
WO2014090985A1
WO2014090985A1 PCT/EP2013/076518 EP2013076518W WO2014090985A1 WO 2014090985 A1 WO2014090985 A1 WO 2014090985A1 EP 2013076518 W EP2013076518 W EP 2013076518W WO 2014090985 A1 WO2014090985 A1 WO 2014090985A1
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seq
single stranded
stranded nucleic
nucleic acid
exon
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PCT/EP2013/076518
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English (en)
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Jana Burkhardt
Holger Kirsten
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Universität Leipzig
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Priority to EP13803055.6A priority Critical patent/EP2931893A1/fr
Publication of WO2014090985A1 publication Critical patent/WO2014090985A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • 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
    • C12N2310/321—2'-O-R Modification
    • 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 present invention relates to the field of biotechnology and medicine. Particularly the present invention relates to single stranded nucleic acids and their use in treating a disease, particularly autoimmune diseases and graft-versus-host disease.
  • a disease particularly autoimmune diseases and graft-versus-host disease.
  • GvHD graft-versus-host disease
  • T-cells A complete depletion of T-cells, however, leads to frequent recurrences of leukemia (B. Glass, M. Nickelsen, P. Dreger, Bone Marrow Transplant. 34(5): 391-7 (2004)).
  • Activation of T-cells occurs through the engagement of both the T-cell receptor complex, including T-cell co receptors, on the T-cell by the major histocompatibility complex (MHC) peptide and B7 family members on the antigen presenting cells (APC), respectively.
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • the T- cell receptor complex is located on the surface of T lymphocytes. It recognizes antigens bound to MHC-molecules (major histocompatibility complex) on antigen presenting cells (APC). The affinity between T-cell receptor is relatively low and unspecific.
  • T-cell activation upon antigen-receptor binding is supported and regulated by complex network of associated enzymes, accessory molecules and co-receptors. Both are required for production of an effective immune response; in the absence of CD28 co- stimulation, T-cell receptor signalling alone results in anergy.
  • the signalling pathways downstream from both CD28 and the T-cell receptor involve many proteins.
  • the co-receptors CD4 and CD8 are distinctive to T-cell subpopulations, either T helper cells or cytotoxic T-cells. Stimulating co-receptors such as CD28 enhance and prolong the T-cell activating signal by interacting with their counterparts on APCs themselves.
  • the T-cell receptor complex exists as a complex of several proteins.
  • the actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCRP) genes.
  • the other proteins in the complex are the CD3 proteins: CD3ey (CD3E and CD3G) and CD3s5 (CD3E and CD3D) heterodimers and, most important, a CD3 ⁇ homodimer, which has a total of six ITAM motifs.
  • the ITAM motifs on the CD3 ⁇ can be phosphorylated by Lck and in turn recruit ZAP-70. Lck and/or ZAP-70 can also phosphorylate the tyrosines on many other molecules, not least CD28, LAT and SLP-76, which allows the aggregation of signalling complexes around these proteins.
  • GvHD GvHD
  • therapeutic stem cell treatment which for example is in principle also a possible treatment of common immunological diseases such as rheumatoid arthritis, but because of the possibility of serious complications it is not widely used in that area.
  • GvHD prophylaxis which comprises a combination of a calcineurin inhibitor (eg. Cyclosporin) and methotrexate (MTX).
  • GvHD prophylaxis include alternative anti-tumor necrosis factor antibodies and manipulation of the graft, such as complete T-cell depletion.
  • First-line treatment options include high-doses of prednisone (S. Paczesny, S. W. Choi, J. L.
  • DMD Duchenne muscular dystrophy
  • AON are used to skipp exons containing frameshift or stop codon mutations, i.e. eliminating mutations which lead to non-functional short and/or aberrant gene products.
  • Van de Vosse utilized oligonucleotides to leap a stop codon in the IL12 receptor gene on the surface of IL12Rbeta deficient T-cells (d. van, V et al., Blood 113, 4548 (2009)).
  • correction of a specific mutation promoted a renewed T-cell function, but T-cell activation was not influenced in a general way.
  • this method suffers from several drawbacks, e.g. off-target/adverse effects, immune stimulation by double stranded nucleic acids.
  • oligonucleotide For exon skipping, in principal two classes of chemically modified oligonucleotide are used: phosphorodiamidate morpholino oligonucleotides (PMOS) such as AVI-4658 (developed by AVI BioPharma, Seattle, USA) and 2'-0-methyl-phosphorothioate oligonucleotides (PS oligomers or PSO) such as PRO051 (Prosensa Therapeutics, Leiden, The Netherlands). PS oligomers are readily water soluble, are sometimes absorbed into cells and little excreted due to their low affinity for plasma proteins. These properties of PS oligomers are largely independent of their sequence and as a class they have a lot of toxicological and pharmacokinetic similarities (F.
  • PMOS phosphorodiamidate morpholino oligonucleotides
  • PS oligomers or PSO 2'-0-methyl-phosphorothioate oligonucleotides
  • PRO051 Pro
  • an agonistic CD28 antibody has failed in clinical studies due to severe side effects of immunological background (induction of a cytokine storm). Presumably, this effect was based on small differences in the spatial conformation of the antigen sequence between humans and experimental animals (B. Schraven, U. Kalinke, Immunity. 28, 591 (2008), G. Suntharalingam et al, N. Engl. J. Med. 355, 1018 (2006)). Moreover, stoichiometric administration is due, as the antibodies may bind only limited amount of receptors simultaneously. Recently, results showed antibody formation in the host against therapeutic antibodies, thus rendering them useless and the treatment becoming ineffective with time.
  • T-cells are also based on gene therapeutics.
  • AON induced exon skipping these either refer to the introduction of complete transcripts which are then to be overexpressed in the target T-cells (e.g. the T-cell inhibitory CTLA4 gene) or a reduced expression of T-cell activating genes (eg. CD28) by siRNA technique (W. Sang et al, Immunol. Lett. 136, 194 (2011)).
  • siRNA technique W. Sang et al, Immunol. Lett. 136, 194 (2011).
  • siRNA technique W. Sang et al, Immunol. Lett. 136, 194 (2011).
  • siRNA technique W. Sang et al, Immunol. Lett. 136, 194 (2011).
  • siRNA and gene transcripts are double stranded oligonucleotides and thus eventually integrate into the genome of host cells, which is the cause for the cancerogenic potential of these methods.
  • Transfection methods for large constructs include transduction by modified viruses (eg. Adenoviruses) which poses a special risk to non-directional integration at an improper location in the recipient genome.
  • modified viruses eg. Adenoviruses
  • virus-induced oncogenesis differs between animal models (mostly murine) and human subjects. Therefore, preclinical testing of the safety of viral-based gene therapy is limited (M. Cavazzana-Calvo, A. Fischer, J. Clin. Invest 117, 1456 (2007)).
  • a permanent integration into the host genome is a prerequisite, for example repression by siRNA treatment of unwanted genes in form of short-hairpin plasmids.
  • RNA interference RNA interference
  • the present invention now provides for single stranded nucleic acids and a method for treating transplants and/or tissues which overcomes the above outlined drawbacks as will be outlined further below.
  • the present invention relates to a single stranded nucleic acid hybridizing to a splicing motif of an exon in a mRNA of a stimulating T-cell receptor or co-receptor, and wherein said exon codes for a transmembrane domain.
  • the single stranded nucleic acid is complementary to said splicing motif.
  • T-cell receptors and co-receptors Stimulating T-cell receptors and co-receptors are known by those of skills in the art.
  • the use of siRNA or antibodies requires stoichiometric administration of the drug, i.e. the amount of siRNA or antibodies has to be precisely calculated to not over or under dose.
  • the need for stoichiometric administration of the active agent is abolished by the provision of a single stranded nucleic acid according to the present invention. Without being bound by theory, this is due to the than expressed soluble form of the respective T-cell receptor or co- receptor after splicing a transmembrane domain.
  • the then expressed soluble receptor or co- receptor blocks activating compounds, like antigens bound to MHC-molecules, and enhances the inhibitory effect.
  • This increases the anti immunogenic effect as it in addition to the reduced amount of expressed complete T-cell receptor or co-receptor and also blocks activating compounds. As exemplified in the Examples of the present application, this allows inhibiting the activation of T-cell response and the resulting expression of cytokines. Thereby, a novel tool to prevent and treat autoimmune diseases is provided by the present invention.
  • the present invention also relates to a method for treating a transplant or tissue comprising the step of contacting the transplant or tissue with a single stranded nucleic acid according to the present invention.
  • the nucleic acid is bound to a magnetic particle and the contacting is performed using magnetofection. It will be appreciated by those of ordinary skill that the contacting is performed to allow transfection of the cell, i.e. the uptake of the single stranded nucleic acid into the cell.
  • the single stranded nucleic acid according to the present invention is bound to a polycation and the contacting is performed using transfection methods, preferably as outlined herein.
  • transfection methods e.g. combinations of magentofection and a polycation, preferably polethylenimine, or combinations of a polycation and liposomal transfection, or combinations of liposomal transfection and magnetofection.
  • the transfection is performed using a combination of magnetofection and a polycation, preferably polyethlenimine, preferably the polyethylenimine has a molecular weight of 10 kDa to 100 kDa, preferably 20 kDa to 30 kDa, more preferably about 25 kDa.
  • the single stranded nucleic acid according to the present invention also provides for further advantages over the substances, compositions and methods for reducing T-cell activity as compared to the prior art.
  • the nucleic acid according to the present invention which allows the inhibition of T-cell response is single stranded, oncogenic effects of double stranded nucleic acids do not occur.
  • siRNAs induce RNAi, i.e. destruction of transcripts and production of further siRNAs covering different sequences of the destroyed mRNA.
  • the induction of RNAi includes a high risk of producing cross-hybridising siRNAs which results in the so called off-target effects.
  • the present invention uses exon skipping rather than RNAi this risk is abolished.
  • the oncogenic potential of the double stranded siRNAs and other double stranded nucleic acid is eliminated by the present invention.
  • stimulating T-cell receptor or co-receptor in context with the present application relates to transmembrane proteins of T-cells involved in the activation of the T-cell.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4 (Gene ID: 920, updated on 5- Aug-2012), CD28 (Gene ID: 940, updated on 5-Aug-2012), CD8A (Gene ID: 925, updated on 5-Aug-2012), CD25 (gene ID: 3559, updated on 5-Aug-2012), CD8B (Gene ID: 926, updated on 5-Aug-2012), CD3D (Gene ID: 915, updated on 5-Aug-2012), CD3E (Gene ID: 916, updated on 5-Aug-2012), CD3G (Gene ID: 917, updated on 5-Aug-2012), CD247 (Gene ED: 919, updated on 5-Aug-2012), , CD45 (PTPRC) (Gene ID: 5788, updated on 5- Aug-2012), ICOS (Gene ID: 29851, updated on l l-Aug-2012
  • the skilled person is able to determine whether a protein is a transmembrane protein. For example he might use commonly known algorithms to detect transmembrane domains in the sequence of a protein. Generally, the presence of transmembrane domains in proteins is determined using hidden Markov model (Krogh et al., Predicting Transmembrane Protein Topology with a Hidden Markov Model: Application to Complete Genomes. J. Mol. Biol. 305:567-580, 2001; Sonnhammer et al., A Hidden Markov Model for predicting transmembrane helices in protein sequences. In J. Glasgow et al., eds.: Proc. Sixth Int. Conf. on Intelligent Systems for Molecular Biology, pages 175-182. AAAI Press, 1998).
  • An example for such an algorithm is the algorithm of the TMHMM Server (http://www.cbs.dtu.dk/services/TMHMM/).
  • the skilled artisan is able to design single stranded nucleic acids being complementary to a splicing motif of an exon in a mRNA of a stimulating T-cell receptor or co-receptor.
  • the skilled artisan can perform a search for transmembrane domains and the splicing motif for the respective exon.
  • the inventors found a method for searching for suited sequences (target sequence) in a stimulating T-cell receptor or co-receptor.
  • the present invention also provides for a method of designing a single stranded nucleic acid according to the present invention comprising the following steps:
  • step (ii) of the method of designing a single stranded nucleic acid according to the present invention comprises the one or more of the following steps:
  • Steps (a) to (e) are each optional.
  • step (ii) of the method for designing a single stranded nucleic acid comprises all steps (a) through (e).
  • Preferred embodiments of the steps are given below. Determination of exons encoding for a transmembrane domain is known by the skilled person. However, in a preferred embodiment the position of an exon encoding the transmembrane domain of the receptor protein is performed using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/; see above). Splicing motifs are known by those of skills in the art (F.O. Desmet, C.
  • splicing motifs are determined by using the freely available software HSF (http://www.umd.be/HSF/ human splicing finder).
  • sections of the target exon are selected, which contain an accumulation of splicing motifs, preferably at the beginning or the end of an exon and containing at least one splice site with a probability of at least 60%, preferably more than 1 splice site with a probability of at least 60%, more preferably more than 2 splice sites with a probability of at least 70%, even more preferably more than 3 splice sites with a probability of at least 70%, per exon.
  • “Uniqueness” in context with the present invention means that the determined target sequence does not occur at other positions in the genome and/or transcriptome.
  • the target sequence does not have 70 % or more identity over a length of 25 bases or more to other sequences within the genome or transcriptome, preferably not 80 % or more, more preferably 90 % or more, very preferably the target sequence does not have 95 % or more identity to other sequences within the genome or transcriptome over a length of 25 bases or more, even more preferred the target sequence does not have 95 % or more identity to other sequences within the genome or transcriptome over a length of 20 bases.
  • the skilled artisan knows tools to determine the identity (also referred to as similarity) of two sequences.
  • a single stranded nucleic acid will be produced as outlined above and will be characterized and validated in vitro for their efficiency of target gene splicing by exon specific PCR, Real time PCR and Western Blot. Reduction of surface T-cell receptor will be monitored by flow cytometry and fluorescence labelled antibodies specific for the T- cell receptor or co-receptor targeted. Such methods are known by those of skills in the art. T-cell function prior and post transfection of functional single stranded nucleic acids may be tested as well. Such tests are commonly known, e.g. T-cells will be stimulated (e.g.
  • CD3E Gene ED 916 Exon 7
  • CD3G Gene ID 917 Exon 1 and Exon 4
  • CD45 PPRC Gene ID: 5788 Exon 15 and Exon 21
  • TRAT1 (TRIM) Gene ID: 50852 Exon 2
  • TCRB (spec. TRBC2) Gene ID: 6957 Exon 4
  • the single stranded nucleic acid hybridizes to a splicing motif of an exon selected form the group consisting of CD4 exon 8, CD 28 exon 3, CD8A exon 4, CD25 exon 7, CD8B exon 4, CD3G exon 1, CD3G exon 4, CD3D exon 3, CD3E exon 7, TCRA (spec. TRAC) exon 3, TCRB (spec.
  • TRBC2 TRBC2
  • the single stranded nucleic acid hybridizes to a splicing motif of an exon selected form the group consisting of CD4 exon 8, CD28 exon 3, CD8A exon 4, and CD25 exon 7.
  • the single stranded nucleic acid hybridizes to a splicing motif of an exon selected from the group consisting of CD4 exon 8, and CD28 exon 3.
  • the single stranded nucleic acid hybridizes to a splicing motif of CD4 exon 8. It will be apperant to the skilled person that the sequence may vary also depending on the individual to whom the nucleic acid shall be applied, or by whom the transplant shall be received.
  • the individual is a mal, preferably selected form the group consisting of human, mice, rats, monkeys, dogs, cats, horses, sheep, and pigs.
  • the single stranded nucleic acid hybridizes to an mRNA sequence (target sequence) selected from the group consisting of SEQ ID NO. 7, SEQ ED NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ BD NO. 120, SEQ ID NO. 121, SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 106, SEQ ID NO. 11 , SEQ ID NO. 12, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 8, SEQ ED NO. 9, and SEQ ID NO.
  • target sequence selected from the group consisting of SEQ ID NO. 7, SEQ ED NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ BD NO. 120, SEQ ID NO. 121, SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 122, SEQ ID NO.
  • T-cell receptor or co receptor being selected from the group consisting of CD4, CD28, CD8A, CD 25, prefereably the single stranded nucleic acid hybridizing to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ED NO. 4, SEQ ED NO. 5, SEQ ID NO. 6, SEQ ED NO. 120, SEQ ED NO.
  • the stimulating T- cell receptor or co receptor is selected from the group consisting of CD4 and CD28, and the single stranded nucleic acid according to the present invention hybridizes to a sequence selected from the group consisting of SEQ ED NO. 7, SEQ ED NO. 4, SEQ ED NO. 5, SEQ ED NO. 6, SEQ ED NO. 120, SEQ ED NO. 121, SEQ ED NO. 1, SEQ ED NO. 2, and SEQ ED NO.
  • the singl stranded nucleic acid hybridizes to a aequence selected from the group consisting of SEQ ED NO. 7, SEQ ED NO. 4, SEQ ED NO. 5, SEQ ED NO. 6, SEQ ED NO. 1, SEQ ED NO. 2, and SEQ ED NO. 3,.
  • the stimulating T-cell receptor or co receptor is CD28, and the single stranded nucleic acid according to the present invention hybridizes to a sequence selected from the group consisting of SEQ ED NO. 1, SEQ ED NO. 2, and SEQ ED NO. 3.
  • the stimulating T-cell receptor or co receptor is CD4, and the single stranded nucleic acid according to the present invention hybridizes to a sequence selected from the group consisting of SEQ ED NO. 7, SEQ ED NO. 4, SEQ ED NO. 5, and SEQ ED NO. 6; preferably SEQ ED NO. 7. It will be apperant that even though the sequences are DNA sequences, they refer to target sequences on mRNA. It will be acknowledged that the single stranded nucleic acid, when hybridizing to the DNA sequence will likewise hybridize to the respective mRNA sequence.
  • the target sequence is a human target sequence, preferably in an exon of a T-cell receptor selected from the group consisting of CD4 exon 8, CD 28 exon 3, CD8A exon 4, CD25 exon 7, CD8B exon 4, CD3G exon 1, CD3G exon 4, CD3D exon 3, CD3E exon 7, TCRA (spec. TRAC) exon 3, TCRB (spec.
  • the target sequence is a human target sequence selected form the group consisting of SEQ ID NO. 7, SEQ ID NO. 106, SEQ ID NO. 120, SEQ ID NO. 121, SEQ ID NO. 122, and SEQ ID NO. 123.
  • hybridization refers to hybridization under stringent conditions in vitro to test sufficient homology of the single stranded nucleic acid to the target sequence. Stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
  • SSC sodium chloride/sodium citrate
  • the single stranded nucleic acid is complementary to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121 , SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 122, SEQ ID NO. 123, and SEQ ID NO. 106.
  • the single stranded neucleic acid is complementary to a sequence selected from the group consisintg of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121, SEQ ID NO. 1, SEQ ID NO.
  • the stimulating T-cell receptor or co receptor is selected from the group consisting of CD28 and CD4, and the single stranded nucleic acid according to the present invention is complementary to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121 SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, , and.
  • the stimulating T-cell receptor or co receptor is CD28, and the single stranded nucleic acid according to the present invention is complementary to a sequence selected from the group consisting of SEQ ED NO. 120, SEQ ID NO. 121 , SEQ ID NO. 1 , SEQ ID NO. 2, and SEQ ID NO. 3, preferably selected from the group consisting of SEQ ID NO. 120 and SEQ ID NO. 121.
  • the stimulating T-cell receptor or co receptor is CD4, and the single stranded nucleic acid according to the present invention is complementary to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, preferably SEQ ID NO. 7.
  • the single stranded nucleic acid comprises a sequence having at least 70 % identity to a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ID NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ED NO. 127, SEQ ED NO. 107, SEQ ED NO. 23, SEQ ED NO. 24, SEQ ED NO. 20, SEQ ED NO. 21 , and SEQ ED NO. 22, preferably selected from the group consisting of SEQ ED NO. 19, SEQ ED NO.
  • the single stranded nucleic acid comprises a sequence having at least 80 % identity to a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO.
  • SEQ ED NO. 127 SEQ ED NO. 107, SEQ ED NO. 23, SEQ ED NO. 24, SEQ ED NO. 20, SEQ ED NO. 21 , and SEQ ED NO. 22, preferably selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ED NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ED NO. 127, and SEQ ED NO.
  • the single stranded nucleic acid comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ID NO. 18, SEQ ED NO. 124, SEQ ID NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ED NO. 127, and SEQ ED NO. 107, SEQ ED NO. 23, SEQ ED NO. 24, SEQ ED NO. 20, SEQ ED NO. 21 , and SEQ ED NO.
  • the single stranded nucleic acid consists of a sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 16, SEQ ED NO. 17, SEQ ID NO. 18, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 20, SEQ ID NO.
  • SEQ ID NO. 21 preferably selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 126, SEQ ID NO. 127, and SEQ ID NO. 107.
  • the stimulating T-cell receptor or co receptor is selected from the group consisting of CD4, andCD28, and the single stranded nucleic having at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15,.
  • the stimulating T-cell receptor or co receptor is selected from the group consisting of CD28 and CD4, and the single stranded nucleic has at least 80 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO.
  • the stimulating T-cell receptor or co receptor is selected from the group consisting of CD4 and CD28, and the single stranded nucleic has at least 90 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ED NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15,.
  • the stimulating T-cell receptor or co receptor is selected from the group consisting of CD4 and CD28, and the single stranded nucleic has at least 95 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co receptor is selected from the group consisting of CD4 and CD28, and the single stranded nucleic has at least 99 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO.
  • the single stranded nucleic acid has a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ID NO. 125,SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co-receptor is CD28
  • the single stranded nucleic has at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO.
  • the stimulating T-cell receptor or co receptor is CD28, and the single stranded nucleic has at least 80 % identity to a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO.
  • the stimulating T-cell receptor or co receptor is CD28, and the single stranded nucleic has at least 90 % identity to a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co receptor is CD28, and the single stranded nucleic has at least 95 % identity to a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co receptor is CD28, and the single stranded nucleic has at least 99 % identity to a sequence selected from the group consisting of SEQ ED NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO.
  • the single stranded nucleic acid has a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co-receptor is CD8A, and the single stranded nucleic has at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO. 126, and SEQ ID NO. 127.
  • the stimulating T-cell receptor or co receptor is CD8A, and the single stranded nucleic has at least 80 % identity to a sequence selected from the group consisting of SEQ ID NO. 126, and SEQ ID NO. 127.
  • the stimulating T-cell receptor or co receptor is CD8A, and the single stranded nucleic has at least 90 % identity to a sequence selected from the group consisting of SEQ ID NO. 126, and SEQ ED NO. 127.
  • the stimulating T-cell receptor or co receptor is CD8A, and the single stranded nucleic has at least 95 % identity to a sequence selected from the group consisting of SEQ ID NO. 126, and SEQ ED NO. 127.
  • the stimulating T-cell receptor or co receptor is CD8A, and the single stranded nucleic has at least 99 % identity to a sequence selected from the group consisting of SEQ ID NO. 126, and SEQ ID NO. 127, preferably the single stranded nucleic acid has a sequence selected from the group consisting of SEQ ID NO. 126, and SEQ ID NO. 127.
  • the stimulating T-cell receptor or co-receptor is CD25, and the single stranded nucleic has at least 70 % identity to SEQ ID NO. 107. In a preferred embodiment the stimulating T-cell receptor or co receptor is CD25, and the single stranded nucleic has at least 80 % identity to SEQ ID NO. 107. In a further preferred embodiment the stimulating T-cell receptor or co receptor is CD25, and the single stranded nucleic has at least 90 % identity to SEQ ID NO. 107. In yet a further preferred embodiment the stimulating T-cell receptor or co receptor is CD25, and the single stranded nucleic has at least 95 % identity to SEQ ID NO. 107.
  • the stimulating T- cell receptor or co receptor is CD25, and the single stranded nucleic has at least 99 % identity to SEQ ED NO. 107, preferably the single stranded nucleic acid has the sequence of SEQ ID NO. 107.
  • the stimulating T-cell receptor or co-receptor is CD4, and the single stranded nucleic has at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.
  • the stimulating T-cell receptor or co receptor is CD4, and the single stranded nucleic has at least 80 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ED NO. 18. In a further preferred embodiment the stimulating T-cell receptor or co receptor is CD4, and the single stranded nucleic has at least 90 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.
  • the stimulating T-cell receptor or co receptor is CD4, and the single stranded nucleic has at least 95 % identity to a sequence selected from the group consisting SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ED NO. 18.
  • the stimulating T-cell receptor or co receptor is CD4, and the single stranded nucleic has at least 99 % identity to a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18, preferably the single stranded nucleic acid has a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.
  • the single stranded nucleic acid according to the present invention is a splice switching oligonucleotide (SSO) which is antisense to the target mRNA, i.e. is an anti-sense oligonucleotide (AON).
  • SSO splice switching oligonucleotide
  • AON anti-sense oligonucleotide
  • the single stranded nucleic acid according to the present invention is essentially incapable of recruiting RNAseH when formed in a duplex with a complex with a complementary mRNA molecule.
  • the skilled person knows tools and methods to determine whether RNAseH is recruited.
  • the single stranded nucleic acid according to the invention does not mediate RNAseH based cleavage of a complementary single stranded RNA molecule.
  • a stretch of at least 5 consecutive DNA nucleobases are required for an oligonucleotide to be effective in recruitment of RNAseH.
  • the single stranded nucleic acid according to the present invention comprises nucleotide analogues as outlined herein below.
  • the single stranded nucleic acid may also comprise DNA nucleobases, preferably 4 or less consecutive DNA nucleobases.
  • EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH.
  • a compound is deemed capable of recruiting RNase H if, when provided with the complementary RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1 %, such as at least 5 %, such as at least 10 % or less than 20 % of the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothiote linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Examples 91 to 95 of EP 1 222 309.
  • a compound is deemed essentially incapable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 20 % such as less than 10 % such as less than 5 %, or preferably less than 1 %, (or even less than 0.1 %) of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Examples 91 to 95 of EP 1 222 309.
  • the nucleic acid is selected from the group consisting of DNA, RNA and nucleotide analogues or combinations thereof.
  • the single stranded nucleic acid according to the present invention may comprise or consist of one or more nucleotide analogues.
  • the nucleotide analogues (X) are independently selected form the group consisting of: 2'-0-alkyl-RNA, 2'-OMe-RNA, 2'-amino-DNA, 2'-fluoro-DNA, LNA, PNA, HNA, INA, and morpholinos.
  • the nucleic acid comprises one or more nucleotide analogues.
  • the single stranded nucleic acid according to the present invention comprises both nucleotide analogues (X) and nucleotides (x).
  • the single stranded nucleic acid does not comprise a region of more than 7 consecutive nucleotide analogue units (X), such as not more than 6, not more than 5, not more than 4, not more than 3, or not more than 2 consecutive nucleotide analogue units (X).
  • the 5' most nucleobase of the single stranded nucleic acid is a nucleotide analogue (X).
  • the 3' most nucleobase of the single stranded nucleic acid is a nucleotide analogue (X).
  • the 3' most and the 5' most nucleobase is a nucleotide analogue.
  • the single stranded nucleic acid comprises or consists of an alternating sequence of nucleotides and nucleobases.
  • the single stranded nucleic acid comprises at least one LNA analogue unit and at least one further nucleotide analogue unit other than LNA.
  • the single stranded oligonucleotide consists of at least one sequence X2X1X2, wherein X ⁇ is LNA and X 2 is a nucleotide analogue other than LNA, such as either a 2'- OMe RNA unit and 2'-fluoro DNA unit.
  • the sequence of the single stranded oligonucleotide consists of alternating Xj and X 2 units.
  • nucleotide analogue are independently selected form the group consisting of 2'-OMe-RNA, 2'-fluoro-DNA, and LNA.
  • nucleotide analogues are locked nucleic acids (LNA).
  • LNA are selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA, and ena-LNA.
  • the single stranded nucleic acid according to the present invention consists only of LNA and DNA. In one embodiment the single stranded nucleic acid according to the present invention consists only of LNA and DNA units. LNA units in the beta-D configuration are preferred, such as beta-D-oxy or beta-D-thio or beta-D-amino. In one embodiment the LNA may be selected from the group consisting of: beta-D-oxy LNA or beta-D-thio LNA or beta-D-amino LNA, ena-LNA, and optionally including the group consisting of alpha- L-oxy LNA or alpha- L-thio LNA or alpha- L-amino LNA.
  • the single stranded nucleic acid comprises the one or more nucleotide analogues, preferably are selected from the group consisting of beta-D-oxy LNA or beta-D- thio LNA or beta-D-amino LNA, ena-LNA, alpha- L-oxy LNA or alpha- L-thio LNA, and alpha- L-amino LNA.
  • nucleotide sequence motif or nucleotide sequence which consists of only nucleotides
  • the single stranded nucleic acids of the invention which are defined by that sequence may comprise a corresponding nucleotide analogues in place of one or more of the nucleotides present in said sequence, such as LNA units or other nucleotide analogues, which raise the duplex stability/Tm of the oligomer/target duplex (i.e. affinity enhancing nucleotide analogues).
  • the nucleotide analogues may enhance the stability of the oligomer in vivo.
  • the nucleotide analogues (X) are independently selected form the group consisting of 2'-0-alkyl-RNA unit, 2'-0-methyl-RNA unit, 2' MOE RNA unit, 2'-amino- DNA unit, 2'-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, ⁇ unit, and morpholinos unit.
  • the nucleotide analogues (X) are 2-O-methyl-RNA units.
  • affinity-enhancing nucleotide analogues in the oligomer can allow the size of the specifically binding oligomer to be reduced, and may also reduce the upper limit to the size of the oligomer before non-specific or aberrant binding takes place.
  • nucleobase sequence of the single stranded nucleic acid is not fully complementary to the corresponding region of the target sequence
  • the oligomer comprises affinity enhancing nucleotide analogues
  • such nucleotide analogues form a complement with their corresponding nucleotide in the target sequence.
  • the single stranded nucleic acid may thus comprise a simple sequence of natural nucleotides -preferably 2 -deoxynucleotides (referred to here generally as “DNA”), but also possibly ribonucleotides (referred to here generally as “R A”) - and it comprises one or more (and possibly consist completely of) nucleotide "analogues”.
  • DNA 2 -deoxynucleotides
  • R A ribonucleotides
  • Nucleotide “analogues” are variants of natural DNA or RNA nucleotides by virtue of modifications in the sugar and/or base and/or phosphate portions.
  • the term “nucleobase” will be used to encompass natural (DNA- or RNA-type) nucleotides as well as such “analogues” thereof.
  • Analogues could in principle be merely “silent” or “equivalent” to the natural nucleotides in the context of the single stranded nucleic acid; i.e. they do not have an effect on the nucleotide the respective position is hybridising to. Such "equivalent” analogues may nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable to storage or manufacturing conditions.
  • the analogues will have a functional effect on the way in which the single stranded nucleic acid works on exon skipping; for example by producing increased binding affinity to the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell.
  • modification of the nucleotide include modifying the sugar moiety to provide a 2'-substituent group or to produce a bridged (locked nucleic acid) structure which enhances binding affinity and probably also provides some increased nuclease resistance; modifying the intemucleotide linkage from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate.
  • LNA one preferred nucleotide analogue is LNA, such as beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, most preferred beta-D-oxy-LNA.
  • a very preferred nucleotide analogue is 2'-0-Me-RNA (2'-0-Methyl-RNA).
  • the 2'-0-Me- RNA may comprise further modifications, e.g. internucleoside linkage groups as will be outlined herein below.
  • the single stranded nucleotide comprises 5 to 8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues.
  • at least one of said nucleotide analogues is a locked nucleic acid (LNA); for example at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or 8, of the nucleotide analogues may be LNA. In some embodiments all the nucleotides analogues may be LNA.
  • the oligonucleotide of the invention may comprise nucleotide analogues which are independently selected from these types of analogue, or may comprise only one type of analogue selected from the three types.
  • the single stranded nucleic acid according to the invention comprises at least one Locked Nucleic Acid (LNA) unit, preferably between 11 to 18 LNA units.
  • LNA Locked Nucleic Acid
  • the single stranded nucleic acid may comprise both beta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, ena- LNA and/or alpha- LNA in either the D-beta or L-alpha configurations or combinations thereof.
  • the single stranded nucleic acid comprises both LNA and DNA units.
  • the combined total of LNA and DNA units is 16 to 30, more preferably 18 to 25, such as 19 to 24 or 20-25, or 20, or, 21, or 22, or 23.
  • the single stranded nucleic acid comprises both LNA and DNA, and that the nucleic acid comprises one stretch of 5 consecutive DNA nucleotides.
  • all the nucleotide analogues are LNA.
  • the single stranded nucleic acid comprises only LNA nucleotide analogues and nucleotides (RNA or DNA, most preferably DNA nucleotides).
  • the single stranded nucleic acid according to the invention comprises at least one 2'-0-methyl-RNA unit, preferably between 11 to 25 2'-0-methyl-RNA unit.
  • the single stranded nucleic acid comprises both 2'-0- methyl-RNA and DNA units.
  • the combined total of 2'-0-methyl-RNA and DNA units is 16 to 30, more preferably 18 to 25, such as 19 to 24 or 20 to 25, or 20, or, 21, or 22, or 23.
  • the single stranded nucleic acid comprises 2'-0- methyl-RNA and DNA, and that the nucleic acid comprises one stretch of 5 consecutive DNA nucleotides. This allows In some embodiments, all the nucleotide analogues are 2'-0- methyl-RNA.
  • the single stranded nucleic acid comprises only 2'- O-methyl-RNA nucleotide analogues and nucleotides (RNA or DNA, most preferably DNA nucleotides, optionally with modified internucleobase linkages, preferably phosphorothioate).
  • RNA or DNA most preferably DNA nucleotides, optionally with modified internucleobase linkages, preferably phosphorothioate.
  • at least one of said nucleotide analogues is 2 -0- methyl-RNA, preferably 1 1 to 23 nucleobase units.
  • nucleoside analogues are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and are of the following formulas:
  • LNA refers to a bicyclic nucleotide analogue, known as "Locked Nucleic Acid”. It may refer to an LNA monomer, or, when used in the context of an "LNA oligonucleotide” or “LNA single stranded nucleic acid” refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues.
  • LNA locked nucleic acids
  • LNA units and methods of their synthesis are described in inter alia WO 99/14226, WO 00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO 03/006475 and WO 03/095467.
  • the LNA unit may also be defined with respect to its chemical formula.
  • an "LNA unit" as used herein, has the chemical structure shown in Formula 1 below:
  • X is selected from the group consisting of O, S and NRH, where R is H or C]-C 4 -alkyl;
  • Y is (-CH 2 ) r , wherein r is an integer of 1 to 4;
  • B is a base of natural or non-natural origin as described above.
  • r is 1 or 2, and in a more preferred embodiment r is 1
  • the LNA used in the oligonucleotide compounds of the invention preferably has the structure of the general formula
  • Z and Z* are independently selected among an intemucleoside 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 single stranded nucleic acid of the invention comprises at least one LNA unit according to any of the formulas
  • Y is -0-, -S-, -NH-, or N(RH); Z and Z* are independently selected among an intemucleoside 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 LNA used in the oligomer of the invention comprises intemucleoside linkages selected from -0-P(0) 2 -0-, -0-P(0,S)-O, -0-P(S) 2 -0-, -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(RH)-0-, 0-PO(OCH 3 )-0-, 0-PO(NRH)-0-, -0-PO(OCH 2 CH 2 S-R)-0-, -0-PO(BH 3 )-0-, -0-PO(NHRH)-0-, -0-P(0) 2 NRH-, -NRH-P(0) 2 -0-, -NRH-CO-0-, where RH is selected form hydrogen and Ci -4 -
  • 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 -C3 ⁇ 4-N(R)- where R is selected from hydrogen and Amino-LNA can be in both beta-D and alpha-L- configuration.
  • Oxy-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 -C3 ⁇ 4-0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
  • LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.
  • the single stranded nucleic acid according to the invention comprises at least one nucleotide analogue, such as Locked Nucleic Acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide analogues, such as Locked Nucleic Acid (LNA) units, preferably between 3 to 13 nucleotide analogues, such as LNA units, such as 4 to 12, nucleotide analogues, such as LNA units, such as 6 to 10 nucleotide analogues, such as LNA units, preferably 7, 8, or 9 nucleotide analogues, such as LNA units.
  • the LNA units comprise at least one beta-D-oxy-LNA unit(s) such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 beta-D-oxy-LNA units.
  • the single stranded nucleic acid of the invention may comprise more than one type of LNA unit.
  • the compound may comprise both beta-D-oxy-LNA, and one or more of the LNA units selected from the group consisting of thio-LNA, amino-LNA, oxy-LNA, ena- LNA and/or alpha-LNA in either the D-beta or L-alpha configurations or combinations thereof.
  • the single stranded nucleic acid may comprise or consist of both nucleotide analogues, such as 2'-0-methyl RNA, LNA units, and DNA units.
  • 2'-0-methyl RNA, LNA and DNA are preferred, but MOE, , and other 2'-substituted analogues and RNA could also be used.
  • Preferred DNA analogues includes DNA analogues where the 2'-H group is substituted with a substitution other than -OH (RNA) e.g.
  • RNA analogues includes RNA analogues which have been modified in its 2'-OH group, e.g. by substitution with a group other than -H (DNA), for example -O-CH 3 (2'-0- methyl-RNA), -0-CH 2 -CH 2 -0-CH 3 , -0-CH 2 -CH 2 -CH 2 -NH 2 , -0-CH 2 -CH 2 -CH 2 -OH or -F.
  • a very preferred RNA analogue is modified in its 2' -OH group by substitution with -O- CH 3 , i.e. 2'-0-methyl RNA.
  • the single stranded nucleic acid of the invention does not comprise any RNA units.
  • High affintiy nucleotide analogues are nucleotide analogues which result in an oligonucleotide which has a higher thermal duplex stability with a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide. This is typically determined by measuring the T m .
  • Nucleotide analogues which increase the T m of the oligomer/target nucleic acid target, as compared to the equivalent nucleotide are preferred (affinity enhancing nucleotide analogues).
  • the oligomers may be capable of hybridising against the target nucleic acid, such as a the target mRNA sequence, to form a duplex with a T m of at least 30°C, such as 37°C, such as at least 40°C, at least 50°C, at least 55°C, or at least 60°C.
  • the Tm is between 30°C and 80°C, such as between 40°C and 70°C.
  • At least 30%, such as at least 33%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 66%, such as at least 70%, such as at least 80%, such as at least 90% of the nucleobases of the oligomer of the invention are nucleotide analogues nucleobases, such as LNA.
  • all of the nucleobases of the oligomer of the invention are nucleotide analogues nucleobases, such as LNA.
  • single stranded nucleic acid which is used interchangeably with the term “oligomer” or “oligonucleotide” (or simply “oligo), refers, in the context of the present invention, to a molecule formed by covalent linkage of two or more nucleobases.
  • oligonucleotide When used in the context of the single stranded nucleic acid of the invention (also referred to as oligonucleotide), the term “oligonucleotide” may have, in one embodiment, for example between 16 to 30 nucleobases, such as between 18 to 25 nucleobases.
  • the length of the oligonucleotides of the invention may vary. Indeed it is considered advantageous to have oligonucleotides longer than 16 nucleotides, preferably at least 18 nucleobases.
  • the single stranded nuceic acid according to the present invention has a length of 18 to 30 nucleobases, preferably 18 to 25 nucleobases.
  • the single stranded nucleic acid according to the present invention has a length of 18 nucleobases. In a further embodiment the single stranded nucleic acid has a length of 19 nucleobases. In another embodiment the single stranded nucleic acid has a length of 20 nucleobases. In yet a further embodiment the single stranded nucleic acid has a length of 21 nucleobases. In one embodiment the single stranded nucleic acid has a length of 22 nucleobases. In an even further embedment the single stranded nucleic acid has a length of 23 nucleobases. Moreover, another embodiment is the single stranded nucleic acid having a length of 24 nucleobases.
  • intemucleoside linkage group refers to a group capable of covalently coupling together two nucleobases, such as between DNA units, between DNA units and nucleotide analogues, between two nucleotide analogues etc.
  • Preferred examples include phosphate, phosphodiester groups and phosphorothioate groups.
  • the intemucleoside linkage may be selected form the group consisting of -0-P(0) 2 -0-, -O- P(0,S)-0-, -0-P(S) 2 -0, -S-P(0) 2 -0, -S-P(CS)-0-, -S-P(S) 2 -0, -0-P(0) 2 -S-,-0-P(CS)-S-, - S-P(0) 2 -S-, -0-PO(RH)-0-, 0-PO(OCH 3 )-0-, -0-PO(NRH)-0-, -0-PO(OCH 2 CH 2 S- )-0-, -0-PO(BH 3 )-0-, -0-PO(NHRH)-0-, -0-P(0) 2 -NRH-, -NRH-P(0) 2 -0-, -NRH-CO-0-, - NRH-CONRH-, -0-CO-0-, -0-CO-NRH
  • sulphur (S) containing intemucleoside linkages as provided above may be preferred.
  • the intemucleoside linkage group is -0-P(0,S)-0- (phosphorothioate).
  • the nucleotide analogue is 2-O-methyl- RNA and the intemucleoside linkage group is -0-P(0,S)-O.
  • Single stranded nucleic acids comprising or consisting of such are also referred to as 2'-0-methyl phosphorothioate oligonucleotides (PSO).
  • the intemucleoside linkage group is phosphate.
  • one or more of the intemucleoside linkage groups of the single stranded nucleic acid according to the present invention are replaced by intemucleoside linkage groups differing from phosphate.
  • the oligonucleotide of the invention is modified in its intemucleoside linkage group structure, i.e. the modified oligonucleotide comprises an intemucleoside linkage group which differs from phosphate.
  • the oligonucleotide according to the present invention comprises at least one intemucleoside linkage group which differs from phosphate.
  • intemucleoside linkage groups which differ from phosphate (-0-P(0)2-0-) according to the present invention are selected from the group consisting of include -0-P(0,S)-O, -OP(S) 2 -0-, -S-P(0) 2 -0-, -S-P(0,S)-0-, -S-P(S) 2 -0-, - 0-P(0) 2 -S-, -0-P(0) 2 -S-, -0-P(0,S)-S-, -S-P(0) 2 -S-, -0-PO(RH)-0-, 0-PO(OCH 3 )-0-, -0-PO(NRH)-0- , -0-PO(OCH 2 CH 2 S-R)-0-, -0- ⁇ ( ⁇ 3 ⁇ 4)-0-, -0-PO(NHRH)-0-, -0-P(0) 2 -NRH-, -NRH- P(0) 2 -0-, -NRH-CO-0-,
  • one or more of the intemucleoside linkage groups of is 0-P(0,S)-0-, preferably all of the intemucleoside linkage groups are 0-P(0,S)-0.
  • a very preferred embodiment is 2'-OMe- RNA with 0-P(0,S)-0- as intemucleoside linkage groups.
  • the intemucleoside linkage group is preferably a phosphorothioate group (-0-P(0,S)-0- ).
  • all intemucleoside linkage groups of the oligonucleotides according to the present invention are phosphorothioate.
  • the single stranded nucleic acid is fully phosphorothiolated - the exception being for therapeutic oligonucleotides for use in the CNS, such as in the brain or spine where phosphorothioation can be toxic, and due to the absence of nucleases, phosphodiester bonds may be used, even between consecutive DNA units.
  • the single stranded nucleic acid comprises alternating LNA and DNA units (Xx) or (xX).
  • the oligomer comprises a motif of alternating LNA followed by 2 DNA units (Xxx), xXx or xxX.
  • at least one of the DNA or non-LNA nucleotide analogue units are replaced with a LNA nucleobase in a position selected from the positions identified as LNA nucleobase units in any one of the embodiments referred to above.
  • the nucleotide analogue units are independently selected form the group consisting of: 2'-0-alkyl-RNA unit, 2 -0- methyl-RNA unit, 2'-amino-DNA unit, T-fluoro-DNA unit, 2'-MOE-RNA unit, LNA unit, PNA unit, HNA unit, ⁇ unit.
  • "X" is an LNA unit.
  • the single stranded nucleic acid comprises alternating 2'-0-methyl-RNA unit and DNA units (Xx) or (xX).
  • the oligomer comprises a motif of alternating 2'-0-methyl-RNA unit followed by 2 DNA units (Xxx), xXx or xxX.
  • the single stranded nucleic acid comprises at least 3 nucleotide analogue units, such as at least 4 nucleotide analogue units, such as at least 5 nucleotide analogue units, such as at least 6 nucleotide analogue units, such as at least 7 nucleotide analogue units, such as at least 8 nucleotide analogue units, such as at least 9 nucleotide analogue units, such as at least 10, such as at least 11 , such as at least 12 nucleotide analogue units.
  • the oligomer comprises at least 3 2 -O-methyl-RNA units, such as at least 4 2 - O-methyl-RNA unit units, such as at least 5 2'-0-methyl-RNA units, such as at least 6 2 -O- methyl-RNA units, such as at least 7 2'-0-methyl-RNA units, such as at least 8 2'-0-methyl- RNA units, such as at least 9 2'-0-methyl-RNA units, such as at least 10 2'-0-methyl-RNA units, such as at least 11 2'-0-methyl-RNA units, such as at least 12 2'-0-methyl-RNA units.
  • nucleotide analogues such as 2'-0- methyl-RNA unit units
  • cytosine or guanine such as between 1 and 10 of the of the nucleotide analogues, such as LNA units
  • cytosine or guanine such as 2, 3, 4, 5, 6, 7, 8, or 9 of the of the nucleotide analogues, such as 2'-0-methyl-RNA units
  • at least two of the nucleotide analogues are either cytosine or guanine.
  • at least three of the nucleotide analogues are either cytosine or guanine.
  • nucleotide analogues are either cytosine or guanine. In one embodiment at least five of the nucleotide analogues are either cytosine or guanine. In one embodiment at least six of the nucleotide analogues are either cytosine or guanine. Bi one embodiment at least seven of the nucleotide analogues are either cytosine or guanine. In one embodiment at least eight of the nucleotide analogues are either cytosine or guanine. In a preferred embodiment the nucleotide analogues have a higher thermal duplex stability a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide to said complementary RNA nucleotide.
  • the nucleotide analogues confer enhanced serum stability to the single stranded oligonucleotide.
  • the first nucleobase of the single stranded nucleic acid according to the invention, counting from the 3' end is a nucleotide analogue, such as an 2'-0-methyl-RNA unit or LNA unit.
  • the second nucleobase of the single stranded nucleic acid according to the invention, counting from the 3' end is a nucleotide analogue, such as an LNA unit.
  • x denotes a DNA unit.
  • the single stranded nucleic acid has a nucleotide analogue, such as an LNA unit, at the 5' end.
  • all the nucleobases of the single stranded nucleic acid of the invention are nucleotide analogue units.
  • the single stranded nucleic acid according to the invention does not comprise a region of more than 5 consecutive DNA nucleotide units. In one embodiment, the single stranded nucleic acid according to the invention does not comprise a region of more than 6 consecutive DNA nucleotide units. In one embodiment, the single stranded nucleic acid according to the invention does not comprise a region of more than 7 consecutive DNA nucleotide units. In one embodiment, the single stranded nucleic acid according to the invention does not comprise a region of more than 8 consecutive DNA nucleotide units. In one embodiment, the single stranded nucleic acid according to the invention does not comprise a region of more than 3 consecutive DNA nucleotide units.
  • the single stranded nucleic acid according to the invention does not comprise a region of more than 2 consecutive DNA nucleotides.
  • the single stranded nucleic acid comprises a region of 5 to 10 consecutive DNA nucleotides, wherein said region is at an internal position of the single stranded nucleotide, and wherein the other nucleobases are 2'-OMe-RNA, preferably 2'-0-methyl- phophorothioate nucleotides.
  • the single stranded nucleic acid comprises at least two consecutive nucleotide analogue units, such as at least two consecutive 2'-OMe-RNA units. In one embodiment, the single stranded nucleic acid comprises at least region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive 2'-OMe-RNA units. In one embodiment, the single stranded nucleic acid of the invention does not comprise a region of more than 7 consecutive nucleotide analogue units, such as 2'-OMe- RNA units.
  • the single stranded nucleic acid of the invention does not comprise a region of more than 8 consecutive nucleotide analogue units, such as 2'-OMe- RNA units. In one embodiment, the single stranded nucleic acid of the invention does not comprise a region of more than 5 consecutive nucleotide analogue units, such as 2'-OMe- RNA units. In one embodiment, the single stranded nucleic acid of the invention does not comprise a region of more than 4 consecutive nucleotide analogue units, such as 2'-OMe- RNA units.
  • the single stranded nucleic acid of the invention does not comprise a region of more than 3 consecutive nucleotide analogue units, such as 2'-OMe- RNA units. In one embodiment, the single stranded nucleic acid of the invention does not comprise a region of more than 2 consecutive nucleotide analogue units, such as 2'-OMe- RNA units.
  • the nucleobase units of the single stranded nucleic acid of the invention comprises at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or 100 % of nucleotide analogues, such as a Locked Nucleic Acid (LNA) nucleobase and 2'-OMe-RNA.
  • LNA Locked Nucleic Acid
  • the oligonucleotides according to the invention may, in one embodiment, have a sequence selected from the group consisting of the following motifs: an 2'-OMe-RNA nucleobase at every second position of the single stranded nucleic acid, 2'- OMe-RNA nucleobases at every third position of the single stranded nucleotide, 2'-OMe- RNA nucleobases at every position of the single stranded nucleic acid.
  • the single stranded nucleic acid comprises a motif selected from the group consisting of LxLxxLLxxLL, LxLxLLLxxLL, LxxLxxLxxL, xLxxLxxLxx 'Every third', xxLxxLxxLx 'Every third', xLxLxLxLxL 'Every second', LXLXLXL 'Every second', Ld Ld d LLd d LL, LdLdLLLddLLL, LMLMMLLMMLL, LMLMLLLMMLL, LFLFFLLFFLL, LFLFLLLFFLLL, LLLL, LLLLLLL, LLLLLLLL, LLLLLLLLLLL, LLLLLLLLLLL, LLLLLLLLLL, LLLLLLLLLL, LMMLMMLMML, MLMMLMMLMM 'Every third', MMLMMLMMLM 'Every third', MMLMMLMMLM 'Every third', LFFLFFLFFL 'Every third
  • the invention further provides for a single stranded nucleic acid wherein said single stranded nucleic acid comprises either at least one phosphorothioate linkage and/or at least one 3' terminal LNA unit, and/or at least one 5' terminal LNA unit.
  • the single stranded nucleic acid comprises at least one nucleotide analogue selected from the group consisting of LNA and 2'-0-methyl- RNA, preferably 2'0-methyl-RNA with phosphorothioate linkage groups; and the single stranded nucleic acid hybridizes to a splicing motif of an exon selected form the group consisting of CD28 exon 3, CD4 exon 8, CD3G exon 1, CD3G exon 4, CD3D exon 3, CD3E exon 7, TCRA (spec. TRAC) exon 3, TCRB (spec.
  • TRBC2 exon 4
  • CD247 exon 7 CD40LG exon 1, ICOS, exon 3, CD8A exon 4, CD8B exon 4, CD45 (PTPRC) exon 15, CD45 (PTPRC) exon 21, CD25 exon 7, CD122 exon 7, CD132 exon6, ICAM1 exon 7, LAT exon 2 and TRAT1 (TRIM) exon 2; preferably selected from the group consisting of CD28 exon 3, CD4 exon 8, CD3D exon 3, and CD3E exon 7.
  • the single stranded nucleic acid comprises at least one nucleotide analogue selected from the group consisting of LNA and 2'-0-methyl-RNA, preferably 2'0-methyl-RNA with phosphorothioate linkage groups; and hybridizes to a splicing motif of an exon selected form the group consisting of CD28 exon 3, CD4 exon 8.
  • the single stranded nucleic comprises at least one nucleotide analogue selected from the group consisting of LNA and 2'-0-methyl-RNA, preferably 2'0-methyl-RNA with phosphorothioate linkage groups; and acid hybridises to a splicing motif of CD4 exon 8.
  • the single stranded nucleic acid consists of nucleotide analogues selected from the group consisting of LNA and 2'-0-methyl-RNA, preferably 2'O-methyl-RNA with phosphorothioate linkage groups (2'-0-methyl phosphorothioate nucleotides); and the single stranded nucleic acid hybridizes to a splicing motif of an exon selected form the group consisting of CD4 exon 8, CD28 exon 3, CD8A exon 4, CD25 exon 7, CD8B exon 4, CD3G exon 1, CD3G exon 4, CD3D exon 3, CD3E exon 7, TCRA (spec. TRAC) exon 3, TCRB (spec.
  • the single stranded nucleic acid consists of nucleotide analogues selected from the group consisting of LNA and 2'-0-methyl-RNA, preferably 2'O-methyl-RNA with phosphorothioate linkage groups (2'- O-methyl phosphorothioate nucleotides); and hybridizes to a splicing motif of an exon selected form the group consisting of CD4 exon 8, and
  • the single stranded nucleic consists of nucleotide analogues selected from the group consisting of LNA and 2'-0-methyl-RNA, preferably 2'O-methyl-RNA with phosphorothioate linkage groups; and acid hybridizes to a splicing motif of CD4 exon 8.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, CD25, CD3D, and CD3E
  • the single stranded nucleic acid according to the present invention comprises LNA and hybridizes to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ED NO. 4, SEQ ID NO. 5, SEQ ID NO.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, CD25, CD3D, and CD3E, and the single stranded nucleic acid according to the present invention consists of LNA and hybridizes to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO.
  • SEQ ID NO. 6 SEQ ID NO. 120, SEQ ID NO. 121, SEQ ID NO SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 106, SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, CD25, CD3D, and CD3E
  • the single stranded nucleic acid comprises one or more 2'-0-methyl phosphorothioate nucleotides and hybridizes to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121 , SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 106, SEQ ID NO.
  • the single stranded nucleic acid is a 2'-0-methyl phosphorothioate oligonucleotide (PSO) and hybridizes to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121 , SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 122, SEQ ED NO. 123, SEQ ID NO. 106, SEQ ID NO. 11, SEQ ED NO. 12, SEQ ED NO. 8, SEQ ED NO.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, CD25, CD3D, and CD3E, and the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and hybridizes to a sequence selected from the group consisting of SEQ ED NO. 7, SEQ ED NO. 4, SEQ ED NO. 5, SEQ ED NO. 6, SEQ ED NO. 120, SEQ ED NO. 121 , SEQ ID NO. 1, SEQ ED NO. 2, SEQ ED NO. 3, SEQ ID NO. 122, SEQ ED NO. 123, SEQ ED NO. 106, SEQ ED NO. 1 1 , SEQ ED NO. 12, SEQ ED NO. 8, SEQ ID NO. 9, and SEQ ED NO. 10.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid according to the present invention comprises LNA and hybridizes to a sequence selected from the group consisting of SEQ ED NO. 7, SEQ ED NO. 4, SEQ ED NO. 5, SEQ ID NO. 6, SEQ ED NO. 120, SEQ ED NO. 121, SEQ ED NO. 1, SEQ ED NO. 2, SEQ ED NO. 3, SEQ ED NO. 122, SEQ ED NO. 123, and SEQ ED NO. 106.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid according to the present invention consists of LNA and hybridizes to a sequence selected from the group consisting of SEQ ED NO. 7, SEQ ED NO. 4, SEQ ED NO. 5, SEQ ED NO. 6, SEQ ED NO. 120, SEQ ED NO. 121 , SEQ ED NO SEQ ED NO. 1, SEQ ED NO. 2, SEQ ED NO. 3, SEQ ED NO. 122, SEQ ED NO. 123, and SEQ ED NO. 106.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid comprises one or more 2'-0-methyl phosphorothioate nucleotides and hybridizes to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121 , SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 122, SEQ ID NO. 123, and SEQ ID NO.
  • the single stranded nucleic acid is a 2'-0-methyl phosphorothioate oligonucleotide (PSO) and hybridizes to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121 , SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ED NO. 122, SEQ ID NO. 123, and SEQ ID NO. 106.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and hybridizes to a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 120, SEQ ID NO. 121 , SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 122, SEQ ID NO. 123, and SEQ ID NO. 106.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, CD25, CD3D, and CD3E
  • the single stranded nucleic acid according to the present invention comprises one or more LNA and comprises a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ED NO. 18, SEQ ED NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ID NO. 127, SEQ ED NO. 107, SEQ ED NO. 23, SEQ ED NO. 24, SEQ ED NO.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, CD25, CD3D, and CD3E
  • the single stranded nucleic acid according to the present invention consists of LNA and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ED NO. 127, SEQ ED NO.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A,CD25, CD3D, and CD3E, and the single stranded nucleic acid comprises one or more 2'-0-methyl phosphorothioate nucleotides and comprises a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO.
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides, i.e. is a 2'-0-methyl phosphorothioate oligonucleotide (PSO), and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, CD25, CD3D, and CD3E, and the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO.
  • SEQ ED NO. 18 SEQ ED NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ED NO. 127, SEQ ED NO. 107, SEQ ED NO. 23, SEQ ED NO. 24, SEQ ED NO. 20, SEQ ID NO. 21 , and SEQ ED NO. 22.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid according to the present invention comprises one or more LNA and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ED NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ED NO. 127, and SEQ ED NO. 107.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid according to the present invention consists of LNA and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ED NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ID NO. 127, and SEQ ED NO. 107.
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid comprises one or more 2'-0-methyl phosphorothioate nucleotides and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ID NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ID NO. 126, SEQ ED NO. 127, and SEQ ED NO.
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides, i.e. is a 2'-0-methyl phosphorothioate oligonucleotide (PSO), and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ED NO. 127, and SEQ ED NO. 107.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the stimulating T-cell receptor or co-receptor is selected from the group consisting of CD4, CD28, CD8A, and CD25
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15, SEQ ED NO. 126, SEQ ID NO. 127, and SEQ ED NO. 107.
  • the stimulating T-cell receptor or co-receptor is CD4 or CD28
  • the single stranded nucleic acid according to the present invention comprises LNA and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ID NO. 17, SEQ ED NO. 18, SEQ ID NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, and SEQ ED NO. 15, .
  • the stimulating T-cell receptor or co-receptor is CD4 or CD28
  • the single stranded nucleic acid according to the present invention consists of LNA and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO.
  • the stimulating T-cell receptor or co- receptor is CD4 or CD28
  • the single stranded nucleic acid comprises one or more 2'-0- methyl phosphorothioate nucleotides and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ID NO. 18, SEQ ED NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ID NO. 14, and SEQ ID NO.
  • the single stranded nucleic acid is a 2'-0-methyl phosphorothioate oligonucleotide (PSO) and comprises a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ED NO. 16, SEQ ED NO. 17, SEQ ED NO. 18, SEQ ED NO. 124, SEQ ED NO. 125, SEQ ED NO. 13, SEQ ED NO. 14, SEQ ED NO. 15.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the stimulating T-cell receptor or co-receptor is CD4 or CD28
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co-receptor is human CD4 or human CD28
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ID NO. 124, and SEQ ED NO. 125.
  • the stimulating T-cell receptor or co-receptor is a human T-cell receptor or co-receptor.
  • the stimulating T-cell receptor or co-receptor is CD4 or CD28
  • the single stranded nucleic acid according to the present invention comprises LNA and comprises a sequence selected from the group consisting of of SEQ ED NO.
  • the stimulating T-cell receptor or co-receptor is CD4 or CD28
  • the single stranded nucleic acid according to the present invention consists of LNA and comprises a sequence selected from the group consisting of of SEQ ID NO. 19, SEQ ID NO. 124, and SEQ ID NO. 125.
  • the stimulating T-cell receptor or co-receptor is CD4 or CD28
  • the single stranded nucleic acid comprises one or more 2'-0-methyl phosphorothioate nucleotides and comprises a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 124, and SEQ ID NO.
  • the single stranded nucleic acid is a 2'-0-methyl phosphorothioate oligonucleotide (PSO) and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ED NO. 124, and SEQ ED NO. 125.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the stimulating T-cell receptor or co- receptor is CD4 or CD28
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 124, and SEQ ED NO. 125.
  • the stimulating T-cell receptor or co-receptor is CD28
  • the single stranded nucleic acid according to the present invention comprises LNA and comprises a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co-receptor is CD28
  • the single stranded nucleic acid according to the present invention consists of LNA and comprises a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO. 125,SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co-receptor is CD28
  • the single stranded nucleic acid comprises one or more 2'-0-methyl phosphorothioate nucleotides and comprises a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ID NO. 125,SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.
  • the stimulating T-cell receptor or co-receptor is CD28
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has a sequence selected from the group consisting of SEQ ID NO. 124, SEQ ED NO.
  • the T-cell receptor or co-receptor is human CD28 and the single stranded nucleic acid consistis of 2'-0-methyl-phophorothioate nucleotides and has a sequence selected from the group consisting of SEQ ID NO. 124 and SEQ ID NO. 125.
  • the stimulating T-cell receptor or co-receptor is CD4, and the single stranded nucleic acid according to the present invention comprises LNA and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.
  • the stimulating T-cell receptor or co-receptor is CD4, and the single stranded nucleic acid according to the present invention consists of LNA and comprises a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.
  • the stimulating T-cell receptor or co-receptor is CD4, and the single stranded nucleic acid comprises one or more 2 -O-methyl phosphorothioate nucleotides and comprises a sequence selected from the group consisting of SEQ ED NO. 19, SEQ ID NO. 16, SEQ ED NO. 17, and SEQ ID NO. 18, preferably the single stranded nucleic acid is a 2'-0-methyl phosphorothioate oligonucleotide (PSO) and comprises a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ED NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has a sequence selected from the group consisting of SEQ ID NO. 19, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.
  • the T-cell receptor or co-receptor is a human T-cell receptor or co-receptor.
  • the stimulating T-cell receptor or co-receptor is CD4, and the single stranded nucleic acid according to the present invention comprises LNA and comprises the sequence of SEQ ID NO. 19.
  • the stimulating T-cell receptor or co-receptor is CD4, and the single stranded nucleic acid according to the present invention consists of LNA and comprises the sequence of SEQ ID NO. 19.
  • the stimulating T-cell receptor or co-receptor is CD4, and the single stranded nucleic acid comprises one or more 2'-0-methyl phosphorothioate nucleotides and comprises the sequence of SEQ ID NO. 19, preferably the single stranded nucleic acid is a 2'-0-methyl phosphorothioate oligonucleotide (PSO) and comprises the sequence of SEQ ID NO. 19.
  • PSO 2'-0-methyl phosphorothioate oligonucleotide
  • the single stranded nucleic acid consists of 2'-0-methyl phosphorothioate nucleotides and has the sequence of SEQ ID NO. 19.
  • the single stranded nucleic acid according to the present invention is bound to a magnetic particle.
  • the magnetic particles preferably comprise iron oxide.
  • the magnetic particle may be coated with a binding agent.
  • Binding agent in connection with the present application and the magnetic particles refers to molecules or compositions to which the single stranded nucleic acid binds in order to bind the single stranded nucleic acid to the magnetic particle.
  • the magnetic particle is coated with cationic molecules. Such molecules are disclosed in Mykhaylyk O, Zelphati O, Rosenecker J, and Plank C. siRNA delivery by magnetofection; Curr Opin Mol Ther.
  • contacting in connection with the present invention intents to trigger transfection of the so contacted transplant, tissue, cells etc. with the single stranded nucleic acid according to the present invention.
  • Different transfection techniques to cells e.g. human and murine hematopoietic celltypes such as T- cells, may be applied.
  • the skilled artisan knows to determine suited methods in order to achieve high efficiency of transfection by keeping viability of cells after transfection. Transfection efficiency may for example be monitored by transfection of fluorescence labelled oligonucleotides or plasmids containing an eGFP sequence (enhanced green fluorescent protein) to a predetermined amount of cells.
  • eGFP sequence enhanced green fluorescent protein
  • the relative amount of cells showing the fluorescence gives the efficiency in % of cells exhibiting the respective fluorescence.
  • the method chosen shows a transfection efficiency of at least 30 %.
  • Preferred is also a transfection efficiency of at least 40 %, at least 50 %, at least 60 %, or at least 70 %, preferably at least 80 %, more preferably at least 90 %, even more preferred at least 91 %, yet more preferred at least 95 %.
  • the transfected cells exhibit a high viability.
  • a method is preferably chosen that exhibits a viability of at least 50 % of transfected cells, more preferably at least 80 %, even more preferably at least 90 %, yet more preferably about 95 % or more.
  • the preferred method should show a combination of high transfection efficency and high viability, hence the method chosen shows a transfection efficiency of at least 90 % and a viability of at least 90%, preferably a efficiency of at least 95 % and a viability of about 95 % or more, also preferred a transfection efficiency of at least 30 % combined with a desired high viability.
  • the skilled person furthermore may be able to determine the efficiency in down regulating the T-cell receptor cascade of transfected cells in order to determine a suited transfection method.
  • cytokines of transfected cells may for instance be performed by detecting the expression profile of cytokines of transfected cells compared to suited controls.
  • cytokines suited are CD28, CD3D, CD4, EFNG, IL 10, IL2, IL2RA, IL3, IL4, IL6, TNFa, and CSF2.
  • the cytokines downregulated are IL2, IL2RA, IL3, and CSF.
  • the downregulated cytokines are IL10 and IL2, preferably at least IL2. The expression profile of one or more of these may be determined and it is preferred that the cytokines are reduced in their expression after transfection with a single stranded nucleic acid according to the present invention.
  • Suited techniques for detecting the expression profile are known by those of ordinary skills and include real-time PCR and western blot methods.
  • Preferred techniques used for transfection according to the present invention include: liposomal and non- liposomal transfection, electroporation and magnet assisted transfection (magnetofection).
  • contacting of the single stranded nucleic acid is performed with a method selected from the group consisting of magnetofection, lipofection, electroporation and combinations thereof.
  • a particularly preferred combination is magnetofection of liposomes, by which extraordinary good results were reached.
  • Transfection using the polycationic polymer polyethylenimin (PEI) is preferred as well, which is also well suited for combination with magnetic particles, therefore providing an also preferred combined magneto-polycationic transfection.
  • Transfection based on PEI or PEI-magnetic beads is also preferred for in vivo antisense delivery as well as for in vitro and ex vivo methods. Details and preferred embodiments of the methods are outlined herein below.
  • the magnetofection may be performed as follows. The following however is to be seen as exemplary procedure. The skilled person is able to change or adapt different steps and conditions in order to achieve the wanted result.
  • An appropriate amount of cells e.g. 1 x 10 6 cell per cm 2
  • cell binding magnetic beads e.g. Solution S, Promocell, Heidelberg.
  • the bead bound cells are placed in a flat bottom cell culture plate and then onto a strong magnetic plate. The cells will be adhered within minutes and might remain on the plate until the transfection procedure is completed.
  • cells may be cultivated until adherence and formation of a monolayer by methods known in the art.
  • liposomes comprising the single stranded nucleic acid according to the present invention may be formed.
  • AON single stranded nucleic acid
  • a liposomal chemical e.g. 3.6 ⁇ Ibafect® are incubated for 15 min at RT with about 1 g AON.
  • AON-bead complex formation is formed (optional: AON loaded liposome-bead complex). Therefore, an appropriate amount of AON, e.g. 50-1000 ng, is mixed with an appropriate amount of coated magnetic beads (e.g. cationic coated; e.g.
  • ⁇ Matra-A reagent is suitable to transfect 1 ⁇ g AON into JurkaT- cells) and incubated for 15 min at RT in a volume of at least 50 ⁇ medium per 200 ⁇ final cell suspension.
  • the pre-formed AON-bead complex or AON loaded liposome-bead complex is then transferred to the previously adhered cells and the mixture is incubated for at least 15 min at RT.
  • contacting of the single stranded nucleic acid according to the present invention to the cell or transplant is performed using magnetofection.
  • the skilled artisan knows how to contact a single stranded nucleic acid attached to a magnetic particle using magnetofection. Therefore, the magnetic particles comprising the single stranded nucleic acid, optionally in liposomes, are concentrated on the target T-cells or transplant by the influence of an external magnetic field generated by magnets and/or electromagnets.
  • the cellular uptake of the genetic material is accomplished by endocytosis and pinocytosis, two natural biological processes. Consequently, membrane architecture and structure stay intact, in contrast to other physical transfection methods that damage the cell membrane.
  • nucleic acids are then released into the cytoplasm by different mechanisms depending upon the formulation used: 1) is the proton sponge effect caused by cationic polymers coated on the nanoparticles that promote endosome osmotic swelling, disruption of the endosome membrane and intracellular release of DNA form, 2) is the destabilization of endosome by cationic lipids coated on the particles that release the nucleic acid into cells by flip-flop of cell negative lipids and charge neutralization and 3) is the usual viral infection mechanism when virus is used.
  • the biodegradable cationic magnetic nanoparticles are not toxic.
  • the magnetic particles for magnetofection usually have an average diameter of between 50 and 500 nm.
  • the magnetic particles have an average diameter between 50 and 500 nm, preferably between 50 nm and 300 nm, even more preferably between 100 nm and 200 nm.
  • Liposomes are usually small unilamellar or multilamellar vesicles made of cationic, neutral and/or anionic lipids, for example, by ultrasound treatment of liposomal suspensions.
  • the DNA can, for example, be ionically bound to the surface of the liposomes or internally enclosed in the liposome.
  • Suitable lipid mixtures are known in the art and comprise, for example, DOTMA (1, 2-Dioleyloxpropyl-3-trimethylammoniumbromid) and DPOE (Dioleoylphosphatidyl-ethanolamin) which both have been used on a variety of cell lines.
  • transfection since transfection of T-cells is usually not as efficient as in other cell types, magnetofection in combination with lipofection is preferred.
  • transfection is performed using targeted transfection.
  • the magnetic particles or liposomes comprising the single stranded nucleic acid are bound to a ligand targeting T-cells.
  • the particles or liposomes may be bond to a peptide and/or antibody binding a T-cell receptor.
  • Such ligands in one embodiment is selected from the group consisting of the HIV ENV locus consisting of gpl60, gpl20 and gpl4 protein [GenBank: AAC55466.1], or an antibody binding to T-cell (co)receptor, preferably selected from the group consisting of CD4, CD28, CD8 (e.g. CD8A and/or CD8B), CD25, CD3 (e.g. CD3G and/or CD3D and/or CD3E), TCR (e.g. TCRA (spec. TRAC) and/or TCRB (spec.
  • TRBC2 TRBC2
  • CD25 CD122, CD132, CD247, CD40LG, ICOS
  • CD45 PPRC
  • CD45 PPRC
  • ICAM1 LAT and TRAT1
  • TRAT1 TRAT1
  • CD4, CD28, CD8 and CD3 e.g. monoclonal anti human CD3, clone OKT3
  • Examples for targeted transfection are known to the skilled person (Kircheis R, Kichler A, Wallner G, Kursa M, Ogris M, Felzmann T, Buchberger M, Wagner E. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther.
  • Pigment epithelium-derived factor gene loaded in cRGD-PEG-PEI suppresses colorectal cancer growth by targeting endothelial cells.
  • Durymanov MO Beletkaia EA, Ulasov AV, Khramtsov YV, Trusov GA, Rodichenko NS, Slastnikova TA, Vinogradova TV, Uspenskaya NY, Kopantsev EP, Rosenkranz AA, Sverdlov ED, Sobolev AS.
  • the invention also relates to a transgenic cell comprising a single stranded nucleic acid according to the present invention and/or a vector according to the present invention.
  • the transgenic cell according to the present invention is an immune cell, preferably a T-cell.
  • T-cells are known by those of skill in the art as small, non-adhering cells, that belong to a group of white blood cells known as lymphocytes. They are characterized by the presence of a T-cell specific T-cell receptor complex (TCR) on the cell surface, which consists of the TCR-a and TCR- ⁇ chains, CD3 and ⁇ -chain accessory molecules.
  • CD3 is routinely used as marker for T-cells, e.g.
  • the present invention inter alia solves the problem preventing GvHD.
  • the inventors unexpectedly found, that the presence of T-cell receptors or co-receptors on T-cells, e.g. comprised in transplants, may be significantly reduced by the single stranded nucleic acid according to the present invention.
  • the present invention also relates to a transplant comprising immune cells according to the present invention and/or a nucleic acid or a vector according to the present invention.
  • Transplant in connection with the present invention relates to all types of cells, tissues, organs or the like, which are intended to be transplated to a patient.
  • the invention as far as relating to the treatment of transplants, does not relate to surgical transplant procedure itself but merely to the preparation of a transplant in order to prevent immune reactions of the host/recipient against the transplant.
  • the transplant is an allograft, i.e. steming from another individual than the patient to be transplanted with.
  • transplants often comprise several types of cells and tissues.
  • a preferred target of the inventive method and inventive single stranded nucleic acids are T-cells.
  • the term "transplant” is to be understood as the part of the transplant comprising immune cells, preferably T-cells.
  • the transplant is selected from the group consisting of mobilized peripherial stem cells, PMBC, and bone marrow transplant (stem cells), preferably bone marrow stem cells.
  • the transplant preferably is derived from the same species as the host/recipient is.
  • Preferred species are mammals, preferably selected from the group consisting of humans, rodents like mice or rats, monkeys, horse, cats, dogs, sheeps, and pigs, preferably selected from the group consisting of humans and mice, more preferably humans.
  • the invention relates to a single stranded nucleic acid according to the present invention for use as a therapeutic drug. Moreover, the invention also relates to a single stranded nucleic acid according to the present invention as outlined above for use in the treatment of a disease selected from the group consisting of autoimmune diseases (e.g. autoimmune hepatitis, diabetes mellitus type 1, Colitis Ulcerosa, Crohn's disease, Glomerzlonephritis, Psoriasis), inflammatory diseases (e.g. Rheumatoid Athritis, Multiple Sclerosis, Asthma), graft versus host disease (GvHD), and rejection of an allogenic transplant by a patient.
  • autoimmune diseases e.g. autoimmune hepatitis, diabetes mellitus type 1, Colitis Ulcerosa, Crohn's disease, Glomerzlonephritis, Psoriasis
  • inflammatory diseases e.g. Rheumatoid Athriti
  • the disease is graft versus host disease (GvHD)
  • GvHD graft versus host disease
  • the skilled person knows how to administer nucleic acids as a medicament. The skilled person is able to decide on the administration way. For example, if GvHD is to be prevented, the single stranded nucleic acid has to be administered to the graft, e.g. ex vivo by the transfection methods as outlined herein. If however, in the case of an acute outbreak of GvHD or another herein outlined disease is to be treated within a patient, different application forms may be selected to target the area or tissue to be treated in vivo.
  • the skilled artisan is than able to decide whether the tissue in which GvHD is present should be treated by magentofection, lipofection, targeted transfection or combinations thereof by methods outlined herein or known by the skilled persion.
  • the therapeutic drug may be administered accordingly as suspension, pill, or solution.
  • the medicament may be prepared as a solution, e.g. for parenteral application, as a suspension or as a pill.
  • the aim of provision of the single stranded nucleic acid according to the present invention is to allow transfection of target T-cells, e.g. T-cells at the area of the disease.
  • the formulation of the medicament is dependent on the intented application.
  • the single stranded nucleic acid may be provided in a complexed form, e.g. with polyethyleenimine (PEI).
  • magentofection for application in the lung as an aerosol, as described in Symbols P, Gleich B, Flemmer A, Hajek K, Seidl N, Wiekhorst F, Eberbeck D, Bittmann I, Bergemann C, Weyh T, Trahms L, Rosenecker J, Rudolph C; Targeted delivery of magnetic aerosol droplets to the lung. Nat Nanotechnol. 2007;2(8):495-9, which is incorporated herein.
  • the single stranded nucleic acid may further be applied by magnetofection, optionally combined with lipofection, intradermal (ID), intramuscular (EVI), intraosseous (IO), intraperitoneal (IP), intravenous (IV), subcutaneous (SC), intrathecal (IT), or injection into the spinal column or synovial.
  • ID intradermal
  • EVI intramuscular
  • IO intraperitoneal
  • IP intravenous
  • IV subcutaneous
  • injection into the spinal column or synovial or injection into the spinal column or synovial.
  • the application of a magnetic field allows direction of the magnetic particles coated with the single stranded nucleic acid towards the desired region (see e.g. Schwerdt JI, Goya GF, Calatayud MP, Herenu CB, Reggiani PC, and Goya RG. Magnetic field-assisted gene delivery: achievements and therapeutic potential. Curr Gene Ther.
  • transfection of a patient may also be performed by injection, e.g. IV, of the single stranded nucleic acid complexed to PEI as a suspension of nanoparticulates.
  • transfection may also be directed using specific molecules targeting the desired cells, e.g. T- cell antigens binding the surface of T-cells (Lee J, Yun KS, Choi CS, Shin SH, Ban HS, Rhim T, Lee SK, and Lee KY.; T-cell-Specific siRNA Delivery Using Antibody- Conjugated Chitosan Nanoparticles. Bioconjug Chem. 2012 May 31. [Epub ahead of print]; which is incorporated herein by reference).
  • a single stranded nucleic acid according to the present invention can be delivered as is to an individual, a cell, tissue, transplant, graft or organ.
  • a single stranded nucleic acid according to the present invention is dissolved in a solution that is compatible with the delivery method.
  • the solution is a physiological salt solution.
  • Particularly preferred in the invention is the use of an excipient that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell.
  • excipients capable of forming complexes, nanoparticles, micelles, vesicles, magnetic particles, liposomes and/or combinations of the aforementioned that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art.
  • Suitable excipients comprise polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT- 18), lipofectinTM, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver each constitutent as defined herein to a cell.
  • PECs polypropyleneimine or polyethylenimine copolymers
  • SAINT- 18 synthetic amphiphils
  • lipofectinTM lipofectinTM
  • DOTAP synthetic amphiphils
  • viral capsid proteins that are capable of self assembly into particles that can deliver each constitutent as defined herein to a cell.
  • excipients have been shown to efficiently deliver an oligonucleotide such as antisense nucleic acids to a wide variety of cultured cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease
  • Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[l-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release.
  • DOTMA cationic lipid N-[l-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOPE neutral lipid dioleoylphosphatidylethanolamine
  • the neutral component mediates the intracellular release.
  • different lipofection components and compositions may be used, e.g. commercially available lipofection kits like Lipofectamine (life technologies) or IBAfect ( ⁇ GmbH).
  • PEI diethylaminoethylaminoethyl
  • PBCA butylcyanoacrylate
  • PHCA hexylcyanoacrylate
  • PEI Polyethylenimine
  • PEI refers to branched or linear polyethylenimine. It may have different sizes (molecular weights) depending on the application. The skilled artist is able to decide on the respective PEI used.
  • the PEI is branched. It is further preferred that the PEI has a molecular weight between 5 kDa and 100 kDa, preferably between 10 kDa and 50 kDa, even more prefereably between 20 kDa and 30 kDa. . In one particular embodiment the PEI has a molecular weight of about 25 kDa. Even more preferred, the PEI is treatet as published by Werth et al.
  • PEI polyethylenimine
  • the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids.
  • This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self- assembly process to package and mediate intracellular release of an oligonucleotide.
  • the skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an oligonucleotide for use in the current invention to deliver it for the treatment of an inflammatory disorder in humans, preferably treatment of graft versus host disease.
  • a single stranded nucleic acid according to the present invention could be covalently or non-convalently linked to a molecule.
  • a preferred molecule is a ligand as defined below and/or a molecule that alters stability and/or pharmacokinetics and/or pharmacodynamics of said single stranded nucleic acid.
  • stability and/or pharmacokinetics and/or pharmacodynamics could be assessed using assays known to the skilled person.
  • a single stranded nucleic acid according to the present invention could be covalently or non-covalently linked to a ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus.
  • a ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus.
  • Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
  • an oligonucleotide is formulated in a composition or a medicament which is provided with at least an excipient and/or a ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery.
  • the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide and further comprising at least one excipient and/or a ligand for delivery and/or a delivery device of said oligonucleotide to a cell and/or enhancing its intracellular delivery.
  • the invention provides a composition or a preparation which is in the form of a kit of parts comprising an oligonucleotide and a further adjunct compound as later defined herein.
  • the single stranded nucleic acid is bound to magnetic particles and the contacting is performed with magnetofection.
  • the single stranded nucleic acid may furthermore be complexed with polycations as outlined herein above.
  • the invention relates to a single stranded nucleic acid as outlined herein for use in the treatment of graft versus host disease.
  • the single stranded nucleic acid according to the present invention is for use in the treatment of graft versus host disease and the graft versus host disease is treated by contacting the transplant with the single stranded nucleic acid, preferably the single stranded nucleic acid bound to a magnetic particle and the contacting is performed using magnetofection.
  • the invention also relates to a kit comprising the single stranded nucleic acid according to the present invention or a vector according to the present invention.
  • the kit further comprises magnetic particles according to the present invention as outlined above.
  • the present invention also relates to combinations of the single stranded nucleic acids as disclosed herein above.
  • it might be necessary to skip more than one exon e.g. when the stimulating T-cell receptor or co receptor comprises more than one exon encoding for a transmembrane domain.
  • two or more single stranded nucleic acids may be used in order to skip all exons encoding a transmembrane domain.
  • single stranded nucleotides may be combined (e.g.
  • the present invention also relates to a combination of nucleic acids comprising two or more single stranded nucleic acids according to the present invention, wherein said single stranded nucleic acids hybridize to splicing motifs of different exons.
  • a combination of single stranded nucleic acids hybridising to splicing motifs of exons encoding for a transmembrane domain in the mRNA of CD4 and CD28, prefereably hybridising to the splicing motif of exon 8 of CD4 and to the splicing motif of exon 3 of CD28.
  • the embodiments of the above outlined single stranded nucleic acids according to the present invention also apply to the single stranded nucleic aicds fo the combinations according to the present invention. It will be apparent that the methods and components of the present invention as well as the uses as substantially described herein or illustrated in the description and the examples, are also subject of the present invention and claimed herewith.
  • Figure 1 Schematic overview of the method for designing a single stranded nucleic acid according ot the present invention.
  • Figure 2 Transfection with FITC labelled nucleic acids.
  • A human T-cell line, magnification lOx;
  • B human T-cell line, magnification 40x;
  • C murine T-cell line, magnification lOx;
  • D murine T-cell line, magnification 40x.
  • Figure 3 Microscopy of AON-FITC magnetofected cells. Magneto fection (Matra-A; Promocell, Heidelberg) of FITC-labelled scrambled AON (H47) (consisting of 2'-0-Me- RNA with posphorothioate as intemucleoside linkage groups and having the sequence of SEQ ID NO.
  • FIG 4 Transfection rates (A) transfection rate for RLD1, 24 h and 96 h after magnetofection, (B) transfection rate of magnetofection depending on celltype, (C) transfection rate depending on transfection method. For Ibafect+MA enhancer transfection 300 ng AON, for all other methods 500 ng AON were applied per 10 6 cells and incubated for 24 h according to the manufacture's protocol (PromoCell GmbH, Heidelberg). Bars indicate standard deviation.
  • Figure 5 Transfection of oligonucleotide by magnetofection over time. Shown is the transfection of human T-cellline Jurkat with AON-FITC by IBAfect (Promocell, Heidelberg) over 28 days.
  • transfection rate (% of positive transfected cells), intensity of transfected AON (in % of maximum), the absolute count of transfected cells (in % of maximum) and vitality (by trypan blue in %) are shown, fluorescence was measured by flow cytometry.
  • Figure 6 (A) Splicing of CD28 induced by AON aCD28mul. Lanes 1 & 4 show amplification of murine T-cell cDNA, lane 2 & 5 were transfected with 500 ng / 10 6 cells AON aCD28mul (SEQ ID NO. 13) and lane 3 & 6 are controls. Primers had the sequence SEQ ID NO. 87 and SEQ ID NO. 88 for exon 2 to exon 3 and SEQ ID NO. 89 and SEQ ID NO. 90 for exon 2 to exon 4 of the murine CD28 gene, respectively. (B) Exon specific Real- Time PCR: 1.
  • portion of CD28 comprising exon 3 to total CD28 after transfecting murine T-cells with AON aCD28mul.
  • portion of CD4 comprising exon 8 to total CD4 after transfecting murine T-cells with AON aCD4mul (SEQ ID NO. 16).
  • real-time PCR for total cDNA from murine T-cells).
  • FIG. 7 (A) Reduced CD28 T-cell co-receptor expression on the surface of murine T lymphocytes.
  • Murine T lymphocytes (RLD1) were transfected with AON aCD28mul (SEQ ID NO. 1) by magnetic assisted transfection (Matra-A, Promocell). Pre- and post transfection, samples were stained with anti-murine-CD28-PE antibody (BD bioscience) and cellcount as well as fluorescence intensity was measured by FACS analysis (BD canto II);
  • Murine T lymphocytes (RLD1) were transfected with AONs aCD3Dmul (SEQ ID NO. 23), aCD3Dmu2 (SEQ ID NO.
  • aCD3Emul SEQ ID NO. 20
  • aCD3Emu2 SEQ ID NO. 21
  • Pre- and post transfection samples were stained with anti-murine-CD3-PerCP antibody (BD bioscience) and cellcount as well as fluorescence intensity was measured by FACS analysis (BD canto II);
  • C Reduced CD4 T-cell co-receptor expression on the surface of murine T lymphocytes.
  • Murine T lymphocytes (RLD1) were transfected with AON aCD4mul (SEQ ID NO. 16) and aCD4mu2 (SEQ ED NO. 17) by magnetic assisted transfection (Matra-A, Promocell).
  • Pre- and post transfection samples were stained with anti-murine-CD4-APC antibody (BD bioscience) and cellcount as well as fluorescence intensity was measured by FACS analysis (BD canto II).
  • Figure 8 Effect of anti human CD4 AON on CD4 expression in PBMCs.
  • PI lymphocytes containing most CD4+ cells.
  • A shows mean of CD4-APC fluorescence intensity normalized to cell average.
  • B shows CD4+ cell count.
  • C-E show PBMC scatterplots with CD4+ cells outlined with a dotted line and marked by arrow. Decrease of CD4+ cells was observed. Isotype controls were carried along as well (not shown).
  • Figures 9 to 26 Computational analysis of preferred T-cell receptors or T-cell co-receptors. Gene names are given as suggested by HGNC, alternatives are in brackets behind.
  • CCDS consensus sequence were available are given for mRNA, nucleotides and amino acids, underlined sequences alternating with not underlined sequences indicate exons and exon borders, in italics amino acids encoded across a splice junction are highlighted. Geneview as annotated by BLAT algorithm with exons marked thick. TMHMM transmembrane domain prediction results are also given. The resulting amino acid sequences predicted to contain the transmembrane domain are given below the TMHMM prediction analysis. The transmembrane exon is marked by a circle. Preferred variants of the transcripts are marked by a rectangular.
  • Figure 27 Cytokine expression in anti-CD3/anti-CD28 stimulated and unstimulated Jurkat cells transfected wit aCD4hul or a control nucleic acid (mock transfection).
  • Figure 29 Long-term persistence of FAM-labeled antisense oligonucleotides in murin and human immune cell lines.
  • Figure 30 Decrease of murine T-cell surface receptor after transfection with AONs.
  • Figure 31 Decreased proliferation of human peripheral blood mononuclear cells (PBMCs) transfected with function AONs.
  • PBMCs peripheral blood mononuclear cells
  • Cells were transfected with antisense olignucleotide AON1 (aCD4hul, SEQ ID 19) or a combination of AON2 (aCD28hul, SEQ ID SEQ ID NO. 124) and AON3 (aCD28hu2, SEQ ID SEQ ID NO. 125) or siRNA with the same sequence as AON1 (SEQ ID 19) using PEL Cell proliferation was measured using the lyphocyte transformation test.
  • AON1 aCD4hul, SEQ ID 19
  • AON2 aCD28hul, SEQ ID SEQ ID NO. 124
  • AON3 aCD28hu2, SEQ ID SEQ ID NO. 125
  • siRNA siRNA with the same sequence as AON1 (SEQ ID 19) using PEL Cell proliferation was measured using the lyph
  • PBMCs were stimulated by PHA (0.05 ⁇ g ⁇ l) and a-cd3/a- cd28 antibodies (5
  • Cells transfected with scrambled AON (SEQ ID 134) served as a negative control (control).
  • Figure 32 Schematic overview of the murine GvHD model
  • Figure 33 Survival of AON treated animals in a murine GvHD model. Transplants of murine spleen and bone marrow derived from donor strain C57/BL6 were transfected with AON and PEI as outlined herein. Concurrently, host mice (Balb/c) were irradiated with a dose of 0.4 Gy per gram body weight. Thereafter transplantation was conducted by application of the transplant into the caudal vein. The figure shows the days of survival after transplantation. Bars indicated standard deviation of animal groups.
  • control irradiated; no transplant
  • 2 GvHD control (irradiated; untransfected transplant)
  • 3 transfection control (irradiated; transplant transfected with scrambled AON (SEQ ID NO. 95);
  • 4 CD28 (irradiated; transplant transfected with aCD28mul (SEQ ID NO. 13)); 5: CD4 (irradiated; transplant transfected with aCD4mu2) (SEQ ID NO. 17); 6: CD4 and CD28 (irradiated; transplant transfected with aCD4mu2 (SEQ ID NO. 17) and aCD28mul (SEQ ID NO. 13).
  • aCD4mu2 all mice survived until the end of the experiment on day 56.
  • Figure 34 Flow cytometry analysis of host blood shows ablation of cd4+ cells on day 12 and increasing cd4+ rates with donor chimerism on day 40 using donor specific HLA- antibodies (Cat. No. 553570, BD bioscience).
  • FIG 35 Reconstiution of white blood cells (WBC). Hemograms of treated an control animals. For numbering of transplant see figure 33. One mouse of the negative control (3) survived the whole experiment (i.e. all 56 days). This animal exhibited a reconstitution of WBC to the initial value indicating that GvHD was not induced in this animal. Bars indicated standard deviation of animal groups.
  • WBC white blood cells
  • Figure 36 Switch of cd4 + /cd8 + cell ration in murine GvHD. Flow cytometry analysis was performed with the samples indicated. The numbering of the transplant is as in Figure 33 and 35. The analysis shows a reversion of cd4 + /cd8 + ration up to day 12 after transplantation and a recovery of the ration in surviving animals. Bars indicated standard deviation of animal groups.
  • Figure 37 cd4 and cd8 expression in host spleens. Spleen samples were taken from anminals euthanized on day 56. mRNA was isolated and expression of cd4 and cd8 was analyzed as disclosed in the Examples.
  • the figure shows the expression levels in spleen of animals receiving the transplants transfected with either aCD28mul (SEQ ID NO. 13) (1); aCD4mu2 (SEQ ID NO. 17) (2) or cotransfected with aCD28mul (SEQ ID NO. 13) and aCD4mu2 (SEQ ID NO. 17) (3), compared to the expression level in spleens of animals receiving the transplant transfected with the scrambled AON (SEQ ID NO. 134). Bars indicate standard deviation of animal groups. All animals transplanted with a transplant treated according to the present invention showed a significant reduction in cd4 and/or cd8 expression as compared to the control.
  • Figure 38 Decreased gene expression of cytokines in animals treated with CD4-AON compared to mock transfected control. mRNA from animals euthanized on day 56 was extracted and RT-PCR analysis was performed as disclosed herein. Shown is the relative expression level of different cytokines and T-cell receptor target genes in spleens of animals receiving the transplant transfected with aCD4mu2 (SEQ ID NO. 17) compared to the expression level in spleens of animals receiving the transplant transfected with the scrambled AON (SEQ ID NO. 134). Bars indicate standard deviation of animal groups. EXAMPLES
  • Example 1 Selection of suited targets Known stimulating T-cell receptors and co-receptors were selected and suited target sequences were determined using the following procedure according to the present invention: The position of the exons encoding the transmembrane domain of the respective murine or human receptor or co receptor protein were determined using the software of the TMHMM server (http://wwwxbs.dtu.dk/services/TMHMM/). The exon coding for the transmembrane domain splice inducing sequences were determined. In detail, the following steps were done: Sequence segments that contain relevant splicing motifs were determined, using the software "HSF" (http://www.umd.be/HSF/ human splicing finder).
  • AON anti-sense oligonucleotides
  • aCD4mul murine 8 CCA (SEQ ID NO.
  • CD3E 5 ACCUCCACACA
  • CD28 3 UCCACCAACCA
  • CD25 7 GCUGUGUUUUC GATCAGCAGGAAAAC (H2RA) CUGCUGAUC ACAGC (SEQ ID aCD25hul human
  • First transfection efficiency of different methods was determined using FITC or FAM labelled AONs and GFP containing vector plasmids (e.g.: pMax). For this, electroporation (Nucleofection, Lonza, Basel), liposomal and non-liposomal transfection agents (e.g. Lipofectamine, Life technologies, Darmstadt) and magnetic particles assisted transfection (magnetofection) were applied (Promocell, Heidelberg; OZ Bioscience, Kunststoff).
  • electroporation Nucleofection, Lonza, Basel
  • liposomal and non-liposomal transfection agents e.g. Lipofectamine, Life technologies, Darmstadt
  • magnetic particles assisted transfection magnetic particles assisted transfection
  • Transfection rates of up to 95 % for human T-cellline Jurkat and up to 91 % for murine T-cellline RLDl could be reached (by transfection of 0.3 ⁇ g AON, 6 ⁇ IBAfect, 0.3 ⁇ MA Enhancer per 10 6 cells for Jurkat; and 0.5 ⁇ g AON, 0.5 ⁇ Matra-A per 10 6 cells for RLDl, respectively).
  • the highest transfection rate was achieved for human bone marrow at about 94 % (by transfection of 0.5 ⁇ g AON, 0.5 ⁇ Matra-A per 10 6 cells).
  • FIG. 1 illustrates successful transfection of FITC labelled AON in murine and human GVHD relevant tissues and cells by fluorescence microscopy, hi contrast, nucleofection, an electroporation method widely published for application in T-cells (Laforge M, Petit F, Estaquier J, Senik A.
  • Transfection was stable for at least several days and no significant decrease of transfected cells 96 h after magnetofection (figure 4A) or up to 28 d for human T-cellline Jurkat (see Figure 5) was observed. Additionally, a relation between transfection rates and AON amount was found, with amounts as low as 1 ⁇ g and 0.5 ⁇ g AON being similar effective (RLD1 , 10 6 cells, MATra-A, not shown) and transfection rates only decreasing for 0.25 ⁇ g to 0.125 ⁇ g. There is also a celltype and species dependency of magnetic beads to cell count ratio.
  • Figure 4B illustrates celltype dependency of transfection with bone marrow and T-cell lines being most susceptible to magnetofection.
  • figure 4C shows dependency of the transfection efficiency from transfection methods as well as celltypes used.
  • Example 3 Modulating exon skipping and T-cell receptor expression in vitro using AONs
  • Murine T-cells (RLD1 , stimulated with Concanavalin A, ConA) were transfected with AONs for skipping exon 3 of murine CD28 separately or in combination (aCD28mul (SEQ ID NO. 13), aCD28mu2 (SEQ ID NO. 14), and aCD28mu3 (SEQ ID NO. 15); 0.5 ⁇ ⁇ 6 cells).
  • murine T-cells (RLD1, stimulated with ConA) were transfected separately or in combination with AONs for skipping exon 8 of murine CD4 (aCD4mul (SEQ ID NO. 16), aCD4mu2 (SEQ ID NO. 17), and aCD4mu3 (SEQ ID NO.
  • murine T-cells (RLD1, stimulated with ConA) were transfected separately or in combination with AONs for skipping exon 5 of CD3E or exon 3 of CD3D (aCD3Dmul (SEQ ID NO. 23), aCD3Dmu2 (SEQ ID NO. 24), aCD3Emul (SEQ ID NO. 20), aCD3Emu2 (SEQ ID NO. 21); 0.3 ⁇ g/10 6 cells).
  • human PBMCs were transfected with AON for skipping exon 8 of CD4 (aCD4hul (SEQ ID NO. 19), 0.5 ⁇ g/10 6 cells). Transfections were performed using EBAfect (Promocell, Heidelberg) according to the manufacturer's protocol with 6 ⁇ EBAfect agent.
  • primers to amplify exon 2 to exon 3 (cgggaatgggaattttacct (SEQ ED NO.
  • PCRs were performed using standard buffer (2.5 mM MgCl 2 , lx buffer B (solis biodyne, Tartu), 0.3 ⁇ of each primer, 0.2 mM dNTPs and 0.08 U/ ⁇ HotFirePolTaq (solis biodyne, Tartu)).
  • the following program was applied for PCR of murine CD28: 40 rounds, 60°C annealing temperature at, 72°C elongation temperature, and 45 s elongation time at.
  • RT-PCR real-time PCR and FACS analysis were performed.
  • the LightCycler system Roche, Whitneyach
  • lx master mix containing SYBR green and freshly prepared Polymerase
  • 3 mM MgCl 2 containing SYBR green and freshly prepared Polymerase
  • cDNA 50 ng RNA per 20 ⁇ .
  • the cycling program consisted of 20s 95°C (activation), 40 rounds amplification with each round Is at 95°C followed by 20s at 58°C and 20s at 72°C as well as a final melting curve from 72 to 98°C at 0.1 °C / s.
  • Reduction of surface T-cell receptor was monitored by flow cytometry analysis utilizing anti-receptor fluorescence labelled antibodies (human anti CD4-APC, Cat# 555349; murine anti CD28-PE, Cat# 553297; murine anti CD3e-PerCP, Cat# 561089; murine anti CD4- APC, Cat# 561091; all BD bioscience GmbH, Heidelberg according to the manufacturers protocol at 5 ⁇ per 200 ⁇ cell suspension in PBS-PFA).
  • Table 3 shows an overview of T- cell receptor reduction achieved by magnetofection in a ConA stimulated murine T-cell line (RLD1).
  • RLD1 ConA stimulated murine T-cell line
  • Table 3 shows that T-cell receptor presence on the cell surface is significantly reduced after transfection with a single stranded nucleic acid according to the present invention (AON).
  • Table 3 Overview of murine T-cell receptor decrease by AONs in stimulated murine T-cells 24 h post magnetofection
  • Human PBMC cells were transfected with aCD4hul using IBAfect/MA Enhancer at 0.5 ⁇ g AON per 10 6 cells and with 6 ⁇ IBAfect (Promocell, Heidelberg). Briefly, 10 6 cells in 150 ⁇ RPMI 1640 medium with 10 % FKS were mixed with 4.5 ⁇ Solution S and incubated for 15 min at room temperature (RT). Following the incubation time, cells were adhered to the surface of a flat bottom 96 well by 15 min on a magnetic plate (universal plate, Promocell, Heidelberg). Parallel, 0.5 ⁇ g AON in 15 ⁇ serum free Medium X-Vivo was mixed with 6 ⁇ IBAfect and incubated for 20 min at RT.
  • Example 4 Suppression of T-cell activation
  • aCD4hul SEQ ID NO.19
  • Controls were transfected with the scrambled AON of SEQ ED NO. 95 (mock transfection), accordingly.
  • the cells were stimulated with anti-CD3 (Cat# 16-0037-81 ; eBioscience, Frankfurt, Germany) and anti-CD28 antibodies (Cat# 16-0289-81, eBioscience, Frankfurt, Germany).
  • wells of a 96-well microplate were filled with 50 ⁇ of a solution of anti-CD3 antibody (5 ⁇ g/ml) dissolved in PBS.
  • the antibodies were immobilized by incubation at 37°C for 2 h. After washing with PBS (200 ⁇ per well), the transfected Jurkat cells (5 x 10 5 / 160 ⁇ RPMI 1640, 10% FKS) were applied to the wells.
  • CD4-rev (CTTGATGTTGGATTCCAGCAG; SEQ ID NO. 97) for human IL2 detection:
  • IL2-rev (AAGTGAAAGTTTTTGCTTTGAGCTA; SEQ ID NO. 99) for human CD25 (IL2RA) detection
  • IL2RA-rev (CCCGCTTTTTATTCTGCGGAA; SEQ ID NO. 101) for human IL3 detection
  • IL3-rev (GCCCTGTTGAATGCCTCCA; SEQ ID NO. 103)
  • Example 5 Inhibition of Proliferation
  • human peripheral blood mononuclear cells PBMC were transfected with aCD4hul (SEQ ID NO. 19) (AON1), co- transfected with aCD28hul (SEQ ID NO. 124) and aCD28hu2 (SEQ ID NO. 125) (AON 2+3), transfected with an scrambled AON (SEQ ID NO. 134; negative control (Control)), or with a double stranded RNA (dsRNA) having the sequence of aCD4hul (SEQ ID NO. 19) (siRNA).
  • dsRNA double stranded RNA having the sequence of aCD4hul (SEQ ID NO. 19) (siRNA).
  • the dsRNA was not modified, double stranded and served as a positive control.
  • mice C57/BL6 and host mice (Balb/c wt ) were purchased from Charles River (Sulzfeld, Germany). Housing, handling and treating of mice were in accordance with the guidelines of the Universitat Leipzig Anminal Care Committee and the Regional Board of Animal Care for Leipzig (animal experiment registration number TVV53/12 and T75/13).
  • Host mice were irradiated with a dose of 0.4 Gy per g body weight.
  • spleen was removed from donor mice and spleen cells were isolated applying 75 ⁇ cell sieves. Additionally, bonemarrow was flushed out of femur and tibia of both donor legs. Spleen cells and bonemarrow of donors was pooled.
  • Spleen cells were then transfected with AON1 (aCD28mul, SEQ ID 13) or AON2 (aCD4mu2, SEQ ID 17) or scrambled AON (transfection control, SEQ ID 95) or not at all (GvHD control).
  • AON1 aCD28mul, SEQ ID 13
  • AON2 aCD4mu2, SEQ ID 17
  • scrambled AON transfection control, SEQ ID 95
  • GvHD control One groups of hosts was not transplanted (irradiation control).
  • Transfected transplants consisted of 20 mio. spleen cells, transfected with 10 ⁇ g AON and 1 :1 g PEL After formation of the AON-PEI complex for 30 min at room temperature, the complex was added to the spleen cells within 2.5 ml RMPI 1640 media with 10% FCS in a 6- well multidish plate and incubated for additional 4h at 37°C and 5% C0 2 .
  • mice ex vivo application of AONs in a murine GvHD model
  • the general procedure of the animal experiment is outlined in Figure 32. Survival of the mice was monitored over 56 days. The mice were also scored for GvHD phenotype and weighted daily according to Cooke (Cooke K , Hill GR, Crawford JM, Bungard D, Brinson YS, Delmonte J Jr, Ferrara JL (1998) Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J Clin Inv 102(10):1882— 1891). The mice were euthanized at clinical scores of higher than 6, indicating severe GvHD.
  • the blood samples were also analysed for the presence of cd4+ and c8+ positive cells using flow cytometry with cd4 and cd8 specific antibodies (a-cd4-PE, Cat. No. 552775, and a- cd8-PerCP, Cat. No. 553036, BD bioscience).
  • the ratio of cd4+/cd8+ was calculated and results are shown in figure 36.
  • the results demonstrate that the cd4/cd8-ratio flipped at onset of GvHD, but was completely restored in surviving animals.
  • the cd4+/cd8+ ratio was also the most quantitative parameter correlating to clinical phenotype and development of GvHD in individual animals.
  • I l l for IL10: ILlO-for (SEQ ID NO. 112) and ILlO-rev (SEQ ID NO. 113); for IL2: IL2-for (SEQ ID NO. 130) and IL2-rev (SEQ ID NO. 131), for IL2RA (CD25): IL2RA-for (SEQ ID NO. 132) and IL2RA-rev (SEQ ID NO. 133); for IL4: IL4-for (SEQ ID NO. 114) and IL4-rev (SEQ ID NO. 115); for IL6: IL6-for (SEQ ID NO. 116) and IL6-rev (SEQ ID NO.
  • TNFa TNFalpha-for (SEQ ID NO. 118) and TNFalpha-rev (SEQ ID NO. 119).
  • TNFalpha-for SEQ ID NO. 118
  • TNFalpha-rev SEQ ID NO. 119
  • TGTGAGTACTGTGTGGAGGT Preferred target sequence in exon 5 of murine CD3E
  • ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTC human CD28 AATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGT nucleotide sequence CGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTT AGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTT CCGGGC ATC C C TTC AC AAAGGAC TGGA AGTGC TGTGG AAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTT CAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAA ATTGGGC AATGAATC AGTGAC ATTC TACC TC C AGAATT TGTATGTTAACCAAACAGATATTTACTTCTGCAAAATT GAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAA
  • WVLVWGGVLAC YSLLVTVAF 11 transmembrane domain of human CD 28 amino acids 154-176 of SEQ ID NO. 26
  • CD3D variant 1 (amino acids 93-136 of SEQ ID NO. 32) ATGGAACATAGCACGTTTCTCTGGCCTGGTACTGGC human CD3D TACCCTTCTCTCGCAAGTGAGCCCCTTCAAGATACCTA nucleotide sequence TAGAGGAACTTGAGGACAGAGTGTTTGTGAATTGCAAT (Variant 2) ACCAGCATCACATGGGTAGAGGGAACGGTGGGAACACT GCTCAGACATTACAAGACTGGACCTGGGAAAACGCA TCCTGGACCCACGAGGAA A ATAGGTG AATGGGACA GATATATACAAGGACAAAGAATCTACCGTGCAAGTTCA TTATCGAACTGCCGACACACAAGCTCTGTTGAGGAATG ACCAGGTCTATCAGCCCCTCCGAGATCGAGATCGAGATCGAGATGATGCT CAGTACAGCCACCTTGGAGGAAACTGGGCTCGGAACAA GTGA
  • VATIVIVDICITG transmembrane domain of human CD3E amino acids 130-152 of SEQ ID NO. 37
  • LAVLILAIILLQGTLAQSI first transmembrane domain of human CD3G (amino acids 7-25 of SEQ ID NO.
  • transmembrane domain of human CD3G amino acids 115-137 of SEQ ID NO. 40
  • a gene KPKAAEGLDTQRFSGKRLGDTFVL LSDFRRENEGYYF variant 1 unspliced CSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLA GTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGDKP SLSARYV
  • LGLLVAGVLVLLVSLGVAIHLCC transmembrane domain of human CD8B amino acids 173-195 of SEQ ID NO. 52
  • transmembrane domain of human CD45 amino acids 839-861 of SEQ ID NO. 57
  • IFMYLLTVFL transmembrane domain of human CD40LG amino acids 23-45 of SEQ ID NO. 64
  • I VI ITWAAAVIMGTAGLSTYLY transmembrane domain of human ICAM1 amino acids 481-503 of SEQ ID NO. 67
  • TCR2 TCRB
  • CD 132 amino acids 262-284 of SEQ ED NO. 85

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Abstract

La présente invention concerne un acide nucléique à simple brin s'hybridant à un motif d'épissage d'un exon dans un ARNm d'un récepteur ou d'un corécepteur de lymphocytes T stimulateurs, et ledit exon codant pour un domaine transmembranaire. En outre, la présente invention concerne des méthodes de traitement d'une greffe ainsi que des acides nucléiques à simple brin pour l'utilisation en tant que médicament, de préférence pour l'utilisation dans le traitement de la maladie du greffon contre l'hôte.
PCT/EP2013/076518 2012-12-13 2013-12-13 Modulation des lymphocytes t par saut d'exon WO2014090985A1 (fr)

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US20180072789A1 (en) * 2014-12-02 2018-03-15 Roger Williams Hospital Methods and compositions for treating cancer
WO2019004939A1 (fr) * 2017-06-27 2019-01-03 Agency For Science, Technology And Research Oligonucléotides antisens pour moduler la fonction d'un lymphocyte t
WO2022084389A1 (fr) * 2020-10-20 2022-04-28 Immunoa Pte. Ltd. Cellules immunitaires modifiées
US11975025B2 (en) 2019-05-27 2024-05-07 Immatics US, Inc. Viral vectors and use thereof in adoptive cellular therapy

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
US20180072789A1 (en) * 2014-12-02 2018-03-15 Roger Williams Hospital Methods and compositions for treating cancer
WO2019004939A1 (fr) * 2017-06-27 2019-01-03 Agency For Science, Technology And Research Oligonucléotides antisens pour moduler la fonction d'un lymphocyte t
CN111630167A (zh) * 2017-06-27 2020-09-04 新加坡科技研究局 用于调节t细胞功能的反义寡核苷酸
US11975025B2 (en) 2019-05-27 2024-05-07 Immatics US, Inc. Viral vectors and use thereof in adoptive cellular therapy
WO2022084389A1 (fr) * 2020-10-20 2022-04-28 Immunoa Pte. Ltd. Cellules immunitaires modifiées

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