WO1995020317A1 - Procede de preparation de cellules donneuses universelles - Google Patents

Procede de preparation de cellules donneuses universelles Download PDF

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WO1995020317A1
WO1995020317A1 PCT/US1995/001198 US9501198W WO9520317A1 WO 1995020317 A1 WO1995020317 A1 WO 1995020317A1 US 9501198 W US9501198 W US 9501198W WO 9520317 A1 WO9520317 A1 WO 9520317A1
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rnh
oligonucleotide
ifn
cells
ggg
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PCT/US1995/001198
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Marvin R. Garovoy
Bing Huey
Schuman Tam
Robert C. Tam
Murali Ramanathan
Roderick D. Macgregor
C. Anthony Hunt
Marianne Lantz
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The Regents Of The University Of California
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Priority to AU16956/95A priority Critical patent/AU1695695A/en
Publication of WO1995020317A1 publication Critical patent/WO1995020317A1/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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
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    • 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
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • 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
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
    • 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
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3527—Other alkyl chain

Definitions

  • This invention is related to therapeutics, transplantation and immunology. More specifically, it relates to methods for modulating the expression of polypeptides and methods for making cells and organs that are more long lasting when transplanted into a recipient host because of the use of oligonucleotides that reduce the level of transplantation antigens expressed on the cell surface of transplanted cells.
  • Anti-gene code molecules are short R ⁇ A or D ⁇ A transcripts that are "antisense” (i.e., complementary to a R ⁇ A strand in a Watson-Crick pairing manner) to a portion of the normal mR ⁇ A and are not transcribed or translated. Regulation of expression of genes by anti- gene coct ⁇ R ⁇ A, one of the natural modes of gene regulation, was first recognized in prokaryotes (Green, P.M. et al., Ann. Rev. Biochem (1986) 55 . :569) . Natural anti-gene codes and artificial anti-gene codes have been used in prokaryotes to downregulate prokaryotic proteins (Simmons, R.W. et al. , Cell (1983) 34 .
  • TK thymidine kinase
  • Microinjection or transfection of thymidine kinase (TK) anti-gene codes has been shown to inhibit expression of the TK protein (Izant, J.G. et al. , Cell (1984) £6:1007; Kim, S.K. et al. , Cell (1985) 42:129) .
  • short anti-gene codes to the 5' untranslated region of the thymidine kinase gene successfully downregulates protein expression (Izant, J.G. et al., Science (1985) 229:345) .
  • anti-gene codes are the p66 c-src gene (by transfected full length anti-gene codes) , and the c-fos gene [by an anti-gene code spanning the 5' untranslated region of the first exon] (Amini, S. et al. , Mol . Cell. Biol. (1986) 6.:2305; Holt, J.T. et al., Proc. Natl. Acad. Sci. (1986) £:4794) • These anti-gene codes have been introduced under constitutive or heterologous inducible promoters.
  • Synthetic oligomers have also been used to downregulate the expression of c-myc in promyelocytic leukemia cells, and T-lymphocytes (Wickstrom, E.L. et al., Proc. Natl. " Acad. Sci. (1988) 5:1028; Heikkila, R. et al., Nature (1987) 328:445) .
  • C-myc anti-gene code oligonucleotides have been shown to inhibit proliferation in normal hematopoietic cells (Gewirtz, A.M. et al. , Science (1988) 242:1303) .
  • Oligonucleotide-mediated inhibition of gene expression was initially reported by Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA (1978) 75:280. Recent work has demonstrated the effect of oligonucleotides in many model in vi tro systems and has established the potential of using oligonucleotides as a new strategy for treating diseases (Uhlmann, et al . , Chem. Rev. (1990) j__0:544) . It is generally recognized that the principal problems are the susceptibility of oligonucleotides to degradation by nucleases in the biological milieu.
  • oligonucleotides typically have large molecular weights and negative charge densities which do not enable them to readily cross cell membranes and gain cytoplasmic access.
  • Many alternative solutions to these problems have been suggested, making modifications of oligonucleotides in order to impart nuclease resistance.
  • Zon, et al suggests a modification using phosphorothioates (Zon, et al . , in "Oligonucleotides and Analogues - A Practical Approach" (IRL Press, Oxford, 1991), pp. 87-
  • oligonucleotides can improve nuclease resistance but can also result in increased toxicity, reduced binding affinity, and lower activity (Cook 1991, Crooke et al . Anti-Cancer Drug Design (1991) 6.:609-646) .
  • Anti-gene code oligonucleotides may act to prevent transcription by inhibiting RNA polymerase, by binding to mR ⁇ A and preventing splicing or subsequent ribosomal translation, by decreasing the stability of mR ⁇ A through enhancement of mR ⁇ A degradation by R ⁇ ase H, or by preventing or inhibiting the processing to mature mR ⁇ A (Maher, L.J. et al. , Science (1989) 245 :725: Moser, H.E. et al., Science (1987) 238:645; Melton, D.A. et al., Proc. ⁇ atl. Acad. Sci. (1985) £2:144; Gewirtz, A.M.
  • Anti-gene code oligonucleotides may also form a triplex structure with duplex DNA (Moffat, A.S., Science (1991) 152.:1374-1375) . This technique of making anti-gene code oligonucleotides involves the formation of a triplex structure according to certain binding rules.
  • HLA Class II molecules are composed of two non-covalently linked glycoproteins, the a chain and the highly polymorphic ⁇ chain. Each chain contains an extracellular domain, a transmembrane segment and a cytoplasmic tail. The structure of the a. and ⁇ chains and their genes have been elucidated. All known Class II genes are similar in structure and encoded by exons 1 - 4, with exon 5 coding for an untranslated region. The DP, DQ and DR loci all consist of multiple genes. A total of twelve class II genes have been identified. In some haplotypes, some class II genes do not code for a functional peptide and are classified as pseudogenes . Regulation of HLA class II antigen expression by binding anti-gene oligonucleotides to the structural region of the gene has not been reported in the literature.
  • HLA class II antigen expression occurs in part through a series of promoter regions such as the J, W, X (including X x and X 2 ) , and Y boxes, and the IFN- ⁇ response element.
  • IFN- ⁇ is also referred to herein as "gamma interferon” and "IFN ⁇ ”.
  • the X (including X ⁇ and X 2 ) and Y boxes are known to be required in the transcriptional regulation of all class II promoters (Ono, S.J. et al . , Proc. Natl. Acad. Sci . (USA) (1991) ££: 4304-4308) .
  • RF-X Regulatory Factor-X
  • RF-X and its binding site, the X-box are unique and have a high specificity for each other.
  • the DNA binding domain of RF-X consists of 91 amino acids with a basic stretch and shares no notable homology with other known DNA binding motifs (Reith et al . , Genes Dev. (1990) 4 (9) :1528-40) .
  • RF-X binds only the X-box; substitutions in the X-box are generally not well tolerated by RF-X (Hasegawa et al .
  • the X-box sequence is an atypical promoter site, being neither palindromic nor dyad symmetric. Additionally no other sequence (using the program Eugene) shows exact homology with the X-box. No other known cloned transcription factors bind.
  • HLA antigens are implicated in the survival of cell grafts or transplants in host organisms. Although there is acceptable graft survival in the first year for nearly all types of transplants, by five and ten years after transplantation only 40-50% of all grafts are still functioning. This low rate is due to the relentless attack of the immune system on the graft. In addition, death rates of 1-5% are recorded even at the best transplant centers. Drugs are commonly used to control immune responses and prevent graft rejection, and death can be an indirect result of this drug administration.
  • the drugs used to control immune responses usually cause a non-specific depression of the immune system and have toxicity.
  • a patient with a depressed immune system is far more susceptible to life-threatening infections and a variety of neoplasia.
  • the low rate of long term success, and serious risks of infection and cancer are the two main challenges now facing the entire field of tissue and organ transplantation.
  • graft rejection can be prevented or reduced by reducing the levels of exposed HLA antigens on the surface of transplant cells. Faustman, D. et al . , Science (1991) 251:1700-1702, observed that xenograft survival was increased by masking HLA class I surface antigens with F(ab') 2 antibody fragments to HLA class I or tissue specific epitopes
  • This invention contemplates the development of a "universal donor cell" reduced in one or more HLA antigens.
  • the absence of certain HLA antigens on the surface of donor cells, tissues or organs comprising these cells will cause them not to be recognized as foreign and not to elicit a rejection response.
  • By the selective introduction of anti-gene codes into a cell it is possible to block the expression of targeted HLA genes, thereby rendering a graft "invisible" to the immune system.
  • the problem of rejection is eliminated without nonspecific suppression of the immune system, and the immune system remains active to defend against infection and neoplasia.
  • oligonucleotides that reduce the antigenicity of cells are designed to be able to bind in some fashion to a nucleotide sequence relating to a transplantation antigen, and prevent the expression of that antigen.
  • oligonucleotides of the instant invention are designed to be capable of binding to a transplantation antigen nucleotide sequence, it is contemplated that their ultimate mode of action may be different.
  • the present invention gives physicians an improved source of transplantable cells. These transplantation antigen-depleted cells give rise to improved graft survival rates in the recipient or require lower levels of immunosuppressant drug administration in the recipient. These agents and/or the treated cells may also be useful in treating patients with autoimmune diseases.
  • the present invention is drawn to newly discovered classes of chemically modified oligonucleotides and uses thereof. These chemically modified oligonucleotides have been shown to exhibit increased activity and physiological stability and some of them are also capable of interacting with transplantation antigen nucleotide sequences. These modified oligonucleotides may comprise a 3'hydroxyalkylamine, particularly a 3' -CH 2 CH(OH) CH 2 NH 2 group that dramatically increases the physiological stability of the oligonucleotides of the invention. Additionally, these modified oligonucleotides must have one or more phosphodiester internucleotide linkages replaced by a phosphorothioate group.
  • the use of these phosphorothioate linkages is shown to substantially increase activity.
  • the present invention is also drawn to the uses of these modified oligonucleotides including methods for making transplantation antigen-depleted cells, methods of treating autoimmune diseases, and methods of making universal donor organs, as well as the cells and organs produced by these methods.
  • this invention provides methods for making a transplantation antigen-depleted cell from a target cell comprising obtaining the target cell, and then exposing the target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, wherein the oligonucleotide is presented in an amount sufficient to make the target cell a transplantation antigen-depleted cell, and wherein the oligonucleotides has at least one phosphorothioate internucleotide linkage and may have a
  • the oligonucleotide is capable of binding to the nucleotide sequence according to Watson-Crick or triplex binding rules (which includes Hoogsteen-like bonds) .
  • the transplantation antigen is an MHC class I or II antigen.
  • this invention provides methods for making a transplantation antigen-depleted cell from a target cell comprising obtaining the target cell, and then exposing the target cell to an oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen, wherein the oligonucleotide is presented or produced locally in an amount sufficient to make the target cell a transplantation antigen-depleted cell.
  • transplantation antigen-depleted cells prepared by obtaining a target cell, and exposing the target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, wherein the oligonucleotide is presented in an amount sufficient to make the target cell a transplantation antigen-depleted cell, and wherein the oligonucleotide has at least one phosphorothioate internucleotide linkage and may have a 3'hydroxyalkylamine modification.
  • transplantation antigen-depleted cells prepared by obtaining a target cell, and exposing the target cell to an oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen, wherein the oligonucleotide is presented or produced locally in an amount sufficient to make the target cell a transplantation antigen-depleted cell.
  • this invention provides oligonucleotides capable of binding to a transplantation antigen nucleotide sequence through Watson-Crick-type binding or through triplex binding, wherein the oligonucleotides are of use in a method for making a transplantation antigen-depleted cell from a target cell, wherein the oligonucleotide has at least one phosphorothioate internucleotide linkage and may have a 3'hydroxyalkylamine modification.
  • this invention provides oligonucleotides capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen, wherein the oligonucleotide is of use in a method for making a transplantation antigen-depleted cell from a target cell.
  • this invention provides oligonucleotides capable of binding to a transplantation antigen nucleotide sequence through Watson-Crick-type binding or through triplex binding, wherein the oligonucleotides are of use in a method of treating an individual with an autoimmune disease characterized by dysfunctional expression of a transplantation antigen, and wherein the "oligonucleotides capable of binding to a transplantation antigen nucleotide sequence" have at least one phosphorothioate internucleotide linkage and may have a 3'hydroxyalkylamine modification.
  • this inventions provides oligonucleotides capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen, wherein the oligonucleotides are of use in a method of treating an individual with an autoimmune disease characterized by dysfunctional expression of a transplantation antigen.
  • oligonucleotides capable of binding to a double-stranded transplantation antigen nucleotide sequence are provided, wherein the oligonucleotides have at least one phosphorothioate internucleotide linkage and may have a 3'hydroxyalkylamine modification.
  • universal donor organs prepared by obtaining a target organ from an individual, and exposing the target organ to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, wherein the oligonucleotide is presented or produced locally in an amount sufficient to make the target organ a universal donor organ, and wherein the oligonucleotides have at least one phosphorothioate internucleotide linkage and may have a 3'hydroxyalkylamine modification.
  • universal donor organs prepared by obtaining a target organ from an individual, and exposing the target organ to an oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen, wherein the oligonucleotide is presented or produced locally in an amount sufficient to make the target organ a universal donor organ.
  • methods of treating an individual with an autoimmune disease characterized by dysfunctional expression of a transplantation antigen comprising administering to that individual an oligonucleotide capable of binding to a portion of the transplantation antigen nucleotide sequence, in an amount sufficient to inhibit expression of the transplantation antigen, wherein the oligonucleotide has at least one phosphorothioate internucleotide linkage and may have a 3'hydroxyalkylamine modification.
  • the present invention further embodies methods for modulating the expression of a polypeptide, wherein the expression is regulated by IFN- ⁇ , said method comprising, exposing a cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, said oligonucleotide being present in an amount sufficient to disrupt the IFN- ⁇ -regulated expression of said polypeptide in said cell.
  • the present invention embodies methods for modulating the expression of a polypeptide, wherein the expression is regulated by IFN- ⁇ , said method comprising, exposing a cell to an oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen, said oligonucleotide being present in an amount sufficient to disrupt the IFN- ⁇ - regulated expression of said polypeptide in said cell .
  • WO 93/14769 discloses unmodified oligonucleotides capable of binding to a transplantation antigen nucleotide sequence, and uses of such unmodified oligonucleotides including methods for making transplantation antigen- depleted cells, methods of treating autoimmune diseases, and methods of making universal donor organs, as well as the cells and organs produced by these methods.
  • Figs. 1(a) - 1(c) are graphs showing data from flow cytometry expressed as a percentage of the level obtained with IFN- ⁇ in the absence of oligonucleotide.
  • Oligonucleotide I inhibition of MHC-I induction by IFN- ⁇ is dose-dependent.
  • Oligonucleotide II treatment does not inhibit the effects of IFN- ⁇ . Control cells were not treated with IFN- ⁇ or oligonucleotide.
  • Figs. 2(a) - 2(c) are graphs showing data from flow cytometry expressed as a percentage of the level obtained with IFN- ⁇ in the absence of oligonucleotide.
  • Oligonucleotide I inhibition of MHC-I induction by IFN- ⁇ is dose-dependent.
  • Oligonucleotide II treatment does not inhibit the effects of IFN- ⁇ . Control cells were not treated with IFN- ⁇ or oligonucleotide.
  • Figs. 3(a) - 3(c) are graphs showing data from flow cytometry expressed as a percentage of the level obtained with IFN- ⁇ in the absence of oligonucleotide.
  • K562 cells were treated with either — ⁇ — oligonucleotide.
  • Oligonucleotide I inhibition of MHC-I induction by IFN- ⁇ is dose-dependent.
  • Oligonucleotide II treatment does not inhibit the effects of IFN- ⁇ . Control cells were not treated with IFN- ⁇ or oligonucleotide.
  • IFN-/? (6400 U/mL) or IFN-/? (6400 U/mL) .
  • Levels are expressed as a percentage of the appropriate IFN induced value for (A) Control cells (B) Cells treated only with the indicated IFN, and (C) Cells treated with the indicated IFN plus 25 ⁇ M oligonucleotide I. Oligonucleotide I is selective for IFN-. ⁇ and does not inhibit MHC-I induced by either IFN- ⁇ or IFN-3 under these conditions.
  • Figs. 6(a) - 6(c) are histograms showing cell surface ICAM-1 levels 24, 48, and 72 hours after K562 cells were treated with 25 ⁇ M oligonucleotide I and either IFN- ⁇ (800 U/mL) or TNF- ⁇ (800 U/mL) . Levels are expressed as a percentage of the appropriate induced value for (A) Control cells (B) Cells treated with the indicated cytokine, and (C) Cells treated with the indicated cytokine and 25 ⁇ M oligonucleotide I. Oligonucleotide I is selective for IFN- ⁇ under these conditions.
  • Figs. 8(a) - 8(c) are histograms showing data from flow cytometry expressed as a percentage of the level obtained with IFN- ⁇ in the absence of oligonucleotide.
  • the following activity relationship summarizes Figs. 8(a) - 8(c) : Oligonucleotide I « Oligonucleotide III > Oligonucleotide IV «-* ⁇ Oligonucleotide V > Oligonucleotide VI « No treatment.
  • Figs. 9(a) - 9(c) are histograms showing the effect of 4-day treatment with IFN- ⁇ or with the addition of various doses of unmodified T2 (umT2) or phosphorothioate-3'hydroxyalkylamine (sT2a) , 3 'hydroxyalkylamine (T2a) or methylphosphonate (mpT2) analogs of T2 on HLA-DR expression in HeLa (2 separate experiments, 9(a) and 9(b)) and RPE cells 9(c) .
  • umT2 unmodified T2
  • sT2a phosphorothioate-3'hydroxyalkylamine
  • T2a 3 'hydroxyalkylamine
  • mpT2 methylphosphonate
  • Uninduced HLA-DR expression was shown in untreated cells (UT) . All experiments were repeated with three different batches of each oligonucleotide and representative data are shown from one experiment. All data show mean ⁇ SD from four direct immunofluorescence analyses per sample.
  • Fig. 10 is a histogram showing the temporal recovery of IFN- ⁇ -induced HLA-DR expression in HeLa cells.
  • HeLa cells were treated with IFN- ⁇ alone (IFN) or with IFN- ⁇ and 4 ⁇ M sT2a or T2a.
  • Oligonucleotide-treated cells were recultured in oligonucleotide-free medium on Day 4, and all cells were then restimulated with IFN- ⁇ every 48 hours. Uninduced DR expression at the same time points was assessed in untreated cells (UT) . All data show mean ⁇ SD from four direct immunofluorescence analyses per sample.
  • Fig. 11 is half-tone reproduction of an electropherogram that shows the temporal recovery of IFN- ⁇ -induced HLA-DR transcription in HeLa cells.
  • - Total RNA (20 ⁇ g) isolated from treated-cell preparations was analyzed by RNase protection as described in the text.
  • IFN- ⁇ -induced transcription of HLA-DR mRNA is shown following treatment for 4 (lane 3) , 8 (lane 8) and 10 (lane 10) days.
  • the oligonucleotide-mediated effect on IFN- ⁇ -induced transcription of DR mRNA was assessed in cells treated with T2a (day 4 :lane 4 and day 8:lane 6), sT2a (day 4: lane 5, day 8 :lane 7, day 10:lane 11 and day 12: lane 12) , and control oligonucleotides, COla (see Table 4) (day 4: lane 13) and sCOla (see Table 4) (day 4: lane 14) .
  • No DR mRNA was observed in untreated cells (lane 9) .
  • Hybridization of total RNA with a probe for glyceraldehyde-3-phosphate dehydrogenase mRNA (GDP) demonstrated comparable starting amounts for each sample used. Undigested probe for DR and GDP are shown in lane 1 and lane 2 respectively. Lane 15 shows labeled Haelll- digested ⁇ />X174 used as a molecular weight marker.
  • Fig. 12(a) is a histogram showing a comparison of the dose-dependent inhibition of HLA-DR expression by sCOla or COla controls to that resulting from sT2a in IFN- ⁇ -treated HeLa cells.
  • Fig. 12(b) is a histogram showing a comparison of the inhibition of IFN- ⁇ -induced HLA-DR expression in HeLa cells treated with 4 ⁇ M phosphorothioate- 3'hydroxyalkylamine (sT2a, sCOla, sC02a) or 3'hydroxyalkylamine (T2a, COla, C02a, C03a) oligonucleotides. Induction in cells treated for 4 Days with (IFN) or without (UT) IFN- ⁇ are also shown. All data show mean ⁇ SD from four immunofluorescence analyses per sample.
  • Fig. 13 is an electropherogram that shows the stability of various modifications of T2 oligonucleotide in extracellular supernatants (S, left panel) and HeLa cell lysates (L, right panel) .
  • Cells were incubated with labeled umT2, sT2, T2a or sT2a. Time-independent degradation was followed by autoradiography of electropherograms. Undegraded oligonucleotides (CON), molecular weight standards (STD) , 32 P-ATP (N) and free 32 P-orthophosphate (P) were simultaneously analyzed.
  • CON Undegraded oligonucleotides
  • STD molecular weight standards
  • N 32 P-ATP
  • P free 32 P-orthophosphate
  • Figs. 14(a) and (b) are graphs which show the recovery of full-length labeled oligonucleotides following temporal analysis of oligonucleotide degradation in extracellular supernatant (Panel a) and HeLa cell lysates (Panel b) .
  • Cells were incubated for up to 72 hours with 32 P-labeled umT2 ( ⁇ ) , sT2 (A) T2a (o) or sT2a (•) oligonucleotides.
  • a full length oligonucleotide is defined as the fraction of total label eluted through Nickspin columns ( ⁇ 10 mer, which refers to the number of nucleotides in the oligonucleotide, retained in the column) . Results are shown as percentage of initial radioactivity loaded corrected for elution efficiency.
  • Figs. 15(a) and (b) are histograms and electropherograms which show the stability of T2a and sT2a in extracellular supernatants (S) and RPE cells lysates (L) . Cells were incubated for up to 24 hours with labeled T2a (a) or sT2a (b) .
  • Figs. 16(a) and (b) are graphs which show the kinetics of cellular association of labeled oligonucleotides with HeLa (Panel a) or RPE (Panel b) cells.
  • Cells were incubated with 2 ⁇ M sT2a (•) , T2a ( ⁇ ) , sT2 (A) and unmodified, umT2 (D) oligonucleotides. Representative data are shown from one experiment of each study. Results are the mean ⁇ SD from four analyses per sample.
  • Figs. 16(a) and (b) are graphs which show the kinetics of cellular association of labeled oligonucleotides with HeLa (Panel a) or RPE (Panel b) cells.
  • Cells were incubated with 2 ⁇ M sT2a (•) , T2a ( ⁇ ) , sT2 (A) and unmodified, umT2 (D
  • 17(a) through (d) are half-tone reproductions of micrographs which show the intracellular distribution of fluorescein-labeled T2a (17(a) , 17(c)) and sT2a (17(b) , 17(d)) oligonucleotides in RPE cells.
  • Cells were exposed to 20 ⁇ M oligonucleotide for 5 minutes (17(a), 17(b)) and 20 minutes (17(c), 17(d)) prior to washing in oligonucleotide-free medium.
  • Each photograph represents images from confocal laser microscopy. Magnification is x60.
  • Fig. 18 is a half-tone reproduction of a gel containing cellular proteins extracted from cells treated concurrently with IFN- ⁇ and with Oligonucleotide I, showing blocking of stat91 phosphorylation by Oligonucleotide I.
  • Fig. 19 is a half-tone reproduction of a gel containing cellular proteins extracted from cells treated first with Oligonucleotide I and then with IFN- ⁇ , showing that the stat91 phosphorylation blocking effect of Oligonucleotide I can be removed by washing.
  • Fig. 20 is a half-tone reproduction of a gel from an electrophoresis mobility shift assay showing that Oligonucleotide I does not bind directly to IFN- ⁇ .
  • Fig. 21 is a graph which shows the data from flow cytometry expressed as a percentage of the level obtained with IFN- ⁇ in the absence of oligo. K562 cells were analyzed for cell surface ICAM-1 expression 24 hours
  • Fig. 22 are graphs which show the data from flow cytometry expressed as a percentage of the level obtained with IFN- ⁇ in the absence of oligo.
  • K562 cells were analyzed for cell surface MHC Class I and ICAM-1 expression 24 hours after treatment with the indicated doses of I ( ⁇ ) or unmodified oligo I
  • Fig. 23 is a histogram which shows the data from flow cytometry expressed as a percentage of the level obtained with IFN- ⁇ in the absence of oligo.
  • the data are expressed as a percentage of the appropriate induced value for (A) control cells (B) cells treated with the indicated cytokine, and (C) cells treated with the indicated cytokine and 25 ⁇ M oligo I.
  • the data are expressed as a percentage of the
  • Fig. 27 are histograms which show the levels of cell surface (a) MHC Class I (b) ICAM-1 (c) MHC Class II DR (d) transferrin receptor in cells that were stained
  • IFN- ⁇ 800 U/ml
  • 25 ⁇ M oligo I 25 ⁇ M oligo I
  • the data are expressed as a percentage of the corresponding IFN- ⁇ induced value.
  • Fig. 28 are graphs which show the cell surface
  • Fig. 29 is a graph that shows levels of cell surface ICAM-1 expression data presented as a percentage of the IFN- ⁇ induced ICAM-1 level on cells not treated with I. K562 cells were treated (Protocol B from Example
  • FIG. 30 is a graph which demonstrates the binding of 125 I labeled IFN- ⁇ to K562 cells. 2 x 10 6 cells were treated on ice with various concentrations of labeled IFN- ⁇ and either media ( • ) , 10 ⁇ M I ( ⁇ ) , or 10 ⁇ M II ( A ) . After 1 hour, radioactivity in the cell free supernatant and in the cell pellet were measured in a gamma counter.
  • the practice of the present invention encompasses conventional techniques of chemistry, immunology, molecular biology, biochemistry, protein chemistry, and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g.. Oligonucleotide Synthesis (M.J. Gait ed. 1984) ; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins, eds. , 1984) ; Sambrook, Fritsch & ⁇ Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) ; PCR Technology (H.A. Erlich ed. , Stockton Press) ; R.K. Scope, Protein Purification Principles and Practice (Springer- Verlag) ; and the series Methods in Enzymology (S. Colowic and N. Kaplan eds., Academic Press, Inc.) .
  • transplantation antigen is used to refer to antigenic molecules that are expressed on the cell surface of transplanted cells, either at the time of transplantation, or at some point following transplantation. Generally these antigenic molecules are proteins and glycoproteins.
  • the primary transplantation antigens are products of the major histocompatibility complex (MHC) , located on chromosome 6 in humans.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • MHC antigens are divided into MHC class I antigens (in humans, this class includes HLA-A, -B, and -C antigens) and MHC class II antigens (in humans, this class includes HLA-DP, -DQ, and -DR antigens) .
  • Transplantation antigens also include cell surface molecules other than MHC class I and II antigens. These antigens include the following: (1) the ABO antigens involved in blood cell recognition; (2) cell adhesion molecules such as ICAM, which is involved in leukocyte cell-cell recognition; and (3) ⁇ 2 - microglobulin, a polypeptide associated with the 44 kd heavy chain polypeptide that comprises the HLA-I antigens but is not encoded by the MHC complex.
  • transplantation antigen nucleotide sequence refers to nucleotide sequences associated with genes encoding transplantation antigens.
  • Nucleotide sequences associated with genes include the region of the gene encoding the structural product, including intron and exon regions, and regions upstream of the structural gene associated with transcription, transcription initiation, translation initiation, operator and promoter regions, ribosome binding regions, as well as regions downstream of the structural gene, including termination sites.
  • Nucleotide sequences associated with genes also include sequences found on any form of messenger RNA (mRNA) derived from the gene, including the pre-mRNA, spliced mRNA, and polyadenylated mRNA.
  • mRNA messenger RNA
  • transplantation antigen-depleted cell refers to cells that are in some way depleted in the expression of at least one transplantation antigen. This depletion may be manifested by a reduced amount of antigen present on the cell surface. Preferably, at least about 50%, more preferably about 80%, and even more preferably about 90% of the antigen is eliminated at the cell surface. Most preferably, this depletion results in essentially total absence of the antigen at the cell surface.
  • transplantation antigens are not always constitutively expressed on the cell surface. These antigens have their expression increased at some point shortly after transplant. In these cases, the depletion is manifested by a reduced amount of antigen or complete lack of antigen at the cell surface at the post- transplant point of normal increased expression.
  • a transplantation antigen-depleted cell will have at least one of two properties: (1) the cell will survive in the transplant recipient for time periods significantly longer than normal cells; or (2) the cell will survive in the transplant recipient for time periods commensurate to normal or untreated cells, but will require lower doses of immunosuppressive agents to the transplant recipient.
  • oligomers or “oligo- nucleotides” include RNA, D ⁇ A, or R ⁇ A/D ⁇ A hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • Nucleic acids refers to RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • binding refers to an interaction or complexation between an oligonucleotide and a target transplantation antigen nucleotide sequence, mediated through hydrogen bonding or other molecular forces. As used herein, the term “binding" more specifically refers to two types of internucleotide binding mediated through base-base hydrogen bonding.
  • the first type of binding is "Watson-Crick-type" binding interactions in which adenine-thymine (or adenine-uracil) and guanine-cytosine base-pairs are formed through hydrogen bonding between the bases.
  • An example of this type of binding is the binding traditionally associated with the DNA double helix.
  • triplex binding refers to any type of base- base hydrogen bonding of a third oligonucleotide strand with a duplex DNA (or DNA-RNA hybrid) that is already paired in a Watson-Crick manner. Triplex binding is more fully described in PCT Application No. WO 90/15884 (published 27 December 1990) .
  • the third strand is designed to match each A or T iir one of the duplex strands with T, and each C or G with C, and the third strand runs antiparallel to the matched strand.
  • the third strand is designed to match each A or T in one of the duplex strands with T, and each C or G with G, also running antiparallel to the matched strand.
  • Other types of triplex binding rules are described in PCT Application No. WO 90/15884.
  • One or more types of triplex binding may occur for a given oligonucleotide.
  • Hoogsteen-like bonds refers to hydrogen bonding between bases.
  • oligonucleotides may be referred to herein by a "T" followed by a number, e.g. T2. As shown throughout the identity of the oligonucleotide is the same whether the number that follows the T is in normal type or subscripted, e.g. T 2 .
  • oligonucleotides are synthesized that are capable of binding to a transplantation antigen nucleotide sequence.
  • This invention also provides for oligonucleotides that are capable of inhibiting the IFN- ⁇ -induced cell surface expression of transplantation antigens. While there are oligonucleotides disclosed herein that are capable of both binding to a transplantation antigen nucleotide sequence, and inhibiting the IFN- ⁇ -induced cell surface expression of transplantation antigens, oligonucleotides which inhibit the IFN- ⁇ -induced cell surface expression of a transplantation antigen without binding a transplantation antigen nucleotide sequence are specifically contemplated herein.
  • the binding of an oligonucleotide of the invention to a transplantation antigen nucleotide sequence may occur between the oligonucleotide and a single-stranded sequence through Watson-Crick-type binding, or between the oligonucleotide and a duplex sequence through triplex binding. In either case, the binding capability results in a transplantation antigen-depleted cell which has reduced expression of at least one transplantation antigen at some point after transplant. Because it is contemplated that there may be cross reactivity and homology between structural and control regions of various transplantation antigens, it is also contemplated that the transplantation antigens that are ultimately depleted in the treated cell may be different than the antigen whose nucleotide sequence was originally targeted.
  • the DR A promoter region contains a number of subregions known to be specific binding sites for DNA binding proteins, called the J, W, X (including X x and X 2 ) , and Y boxes. Particularly significant are the X and X 2 boxes, as described herein.
  • Other specific target sequences are within the structure gene. In general, a minimum of approximately 5 nucleotides, preferably at least 10 nucleotides, are necessary to effect the necessary binding to a specific target sequence within the targeted gene, wherein the targeted sequence may be within an intron. By targeting the structural gene region, only two target DNA sequences per cell are required to be bound by this oligonucleotide.
  • oligonucleotides shorter than 15 nucleotides may be feasible if the appropriate interaction can be obtained.
  • the oligonucleotides capable of binding to a transplantation antigen nucleotide sequence need to contain the sequence-conferring specificity, but may be extended with flanking regions and otherwise derivatized or modified.
  • the oligonucleotide may be prepared by any known method, including. * synthetic, recombinant, and purification methods, and may be used alone or in combination with other oligonucleotides specific for the same or different target transplantation antigens.
  • the oligonucleotide capable of binding to a transplantation antigen nucleotide sequence may also contain "interior flanking sequences", which are sequences within a binding sequence that are not capable of binding to the target through Watson-Crick or triplex binding rules.
  • the oligonucleotide may comprise two or more binding regions separated by nonbinding interior flanking sequences. It is also contemplated that the binding sequences may contain one or more mismatches that do not conform to the binding rules. These substitutions are contemplated as part of the invention as long as the oligonucleotide retains its binding capability as described herein.
  • the oligonucleotides of the invention usually comprise the naturally-occurring bases, sugars and phosphodiester linkages.
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced by phosphonate groups, phosphate groups, protected by a standard protecting group, or activated to prepare ad ⁇ ditional linkages to additional nucleotides, or may be conjugated to solid supports.
  • the 5' and 3' terminal OH groups are conventionally free but may be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.
  • the 3' terminal OH of an oligonucleotide of the invention may be substituted with a hydroxyalkylamine to form an oligonucleotide which is modified with a "3'hydroxyalkylamine” .
  • the 3' terminal OH of an oligonucleotide of the invention is substituted with a 3' -CH 2 CH(OH) CH 2 NH 2 group.
  • 3' -RNH 2 -containing fragments refers to fragments of the oligonucleotides of the invention, which comprise a 3' -CH 2 CH(OH) CH 2 ⁇ H 2 group. These fragments need not comprise the original 3' nucleotides of the original oligonucleotides, but rather "3' -RNH 2 -containing fragments" may contain any segment of the original oligonucleotide, so long as the 3'-terminal nucleotide of the fragment is linked to a CH 2 CH(OH) CH 2 NH 2 group.
  • One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to embodiments wherein phosphate [P(0)0] is replaced by P(0)S ("thioate” or "phosphorothioate”) , P(S)S
  • each R or R' is an H, or an independently substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in an oligomer need to be identical.
  • internucleotide linkages of "an oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen the following conditions apply.
  • at least one phosphodiester internucleotide linkage of the oligonucleotide is replaced by an internucleotide linkage selected from the group consisting of phosphorothioate and methylphosphonate. More preferably, a majority of the phosphodiester internucleotide linkages of the oligonucleotide are replaced by phosphorothioate groups. Most preferably, all of the phosphodiester internucleotide linkages are replaced by phosphorothioate internucleotide linkages.
  • the oligonucleotide must have at least one of its phosphodiester internucleotide linkages replaced by a phosphorothioate internucleotide linkage.
  • at least a second phosphodiester internucleotide linkage of the oligonucleotide is replaced by an internucleotide linkage selected from the group consisting of phosphorothioate and methylphosphonate.
  • a majority of the phosphodiester internucleotide linkages of the oligonucleotide are replaced by phosphorothioate groups. Most preferably, all of the phosphodiester internucleotide linkages are replaced by phosphorothioate internucleotide linkages.
  • oligonucleotides incorporate analogous forms of purines and pyrimidines.
  • analogous forms of purines and pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents.
  • An exemplary but not exhaustive list includes aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl- uracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxyuracil, 2-methyl- thio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, queosine, 2-thiocyto
  • uracil as a substitute base for thymine in deoxyribonucleic acid (hereinafter referred to as "dU") is considered to be an "analogous" form of pyrimidine in this invention.
  • the oligonucleotides of the invention may contain analogous forms of ribose or deoxyribose sugars that are generally known in the art.
  • An exemplary, but not exhaustive list includes 2' substituted sugars such as 2' -O-methyl-, 2'-0-allyl, 2'-fluoro- or 2'-azido- ribose, carbocyclic sugar analogs, ⁇ -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • the oligonucleotide of the invention may also be amplified by PCR.
  • the PCR method is well known in the art and described in, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202 and Saiki, R.K., et al. , Science (1988)
  • the amplified DNA may then be recovered as DNA or RNA, in the original single- stranded or duplex form, using conventional techniques.
  • Oligonucleotides of the invention can also be derivatized in various ways, as can the oligonucleotides capable of binding to a transplantation antigen nucleotide sequence.
  • the oligonucleotides of the invention will be derivatized by attaching a nuclear localization signal to it to improve targeted delivery to the nucleus.
  • a nuclear localization signal is the heptapeptide PKKKRKV (pro-lys-lys-lys-arg- lys-val) .
  • the oligonucleotide is to be used for separation of the target substance, conventionally the oligonucleotide will be derivatized to a solid support to permit chromatographic separation.
  • the oligonucleo ⁇ tide is to be used to label the target or otherwise attach a detectable moiety to target
  • the oligonucleotide will be derivatized to include a radionuclide, a fluorescent molecule, a chromophore, biotin, or the like. If the oligonucleotide is to be used in specific binding assays, coupling to solid support or detectable label is also desirable. If it is to be used therapeutically, the oligonucleotide may be derivatized to include ligands which provide targeting to specific cellular sites or permit easier transit of cellular barriers, toxic moieties which aid in the therapeutic effect, or enzymatic activities which perform desired functions at the targeted site.
  • the oligonucleotide may be derivatized to attach to the targeted sequence, to crosslink the targeted sequence (e.g., through psoralen crosslinks) , or to alter, modify or delete all or part of the targeted sequence. In this manner, the oligonucleotide may cause a permanent depletion of a transplantation antigen on a cell and its daughter cells.
  • the oligonucleotide sequence may also be included in a suitable expression system that would provide in situ generation of the desired oligonucleotide.
  • a class of oligonucleotides have been identified which inhibit IFN- ⁇ -induced cell surface expression of several transplantation antigens or immune receptors, including, for example, the MHC Class II (MHC- II) molecules, for example HLA-DR and HLA-DP, as well as MHC-I molecules, ICAM-1, and the receptor for the Fc portion of the antibody IgG (FcR ⁇ ) in cultured cells, but which are ineffective in blocking other cytokines, such as IFN- ⁇ , IFN- ⁇ , interleukin-1 (IL-1) and tumor necrosis factor alpha [also referred to herein as "TNF ⁇ ", “TNF-a” and “TNF- ⁇ ”] (Tarn, et al .
  • IFN- ⁇ inhibiting oligonucleotides include, for example, human cervical carcinoma cell line (HeLa) , K562 cells, fibroblast, keratinocyte, and human monocytes. The exact mechanism by which these oligonucleotides impart their effect on the immune receptors is not completely characterized.
  • IFN- ⁇ is released by activated T lymphocytes and acts by modulating the expression of several critical immune response molecules (Rosa, J. Immunol. (1988) 140 . :1660-1664; and Rothlein, et al . , J. Immunol. (1988) 141:1665-1669) .
  • Rosa J. Immunol. (1988) 140 . :1660-1664
  • Rothlein et al . , J. Immunol. (1988) 141:1665-1669
  • MHC major histocompatibility complex
  • ICAM-1 intracellular adhesion molecule-1
  • the inhibition of cell surface expression of MHC-I and of ICAM-1, at least by some of the oligonucleotides in the family, is selective for IFN- ⁇ mediated induction; that is to say, these members do not inhibit MHC-I induction or ICAM-1 induction by comparable doses of either alpha interferon (IFN- ⁇ ) or beta interferon (IFN-3) .
  • IFN- ⁇ alpha interferon
  • IFN-3 beta interferon
  • oligonucleotides or oligodeoxynucleotide were used to identify the oligonucleotide family of the invention:
  • AGG GTT CGG GGC GCC ATG ACG GC -RNH 2 ; CAG CCT TGA GGA TTC CCC AAC TCC G -RNH 2 ; GCC ACG GAG CGA GAC ATC TCC G -RNH 2 ; CAT CTT CTG CCA TTC TGA AGC CGG -RNH 2 ; where R -CH 2 CH(OH) CH 2 -.
  • oligodeoxynucleotide I The inhibition by oligodeoxynucleotide I is dose-dependent, as shown in the Examples. (Note the "oligodeoxynucleotide I” is also referred to herein as “oligonucleotide I", “oligonucleotide I”, “oligodeoxynucleotide I", and "T2a”.)
  • oligodeoxynucleotide I is also referred to herein as “oligonucleotide I", “oligonucleotide I", “oligodeoxynucleotide I", and "T2a”.
  • oligodeoxynucleotide I 5' AC ACA ACA CCC AAC ACA ACC AAC CCC-RNH 2 was inactive.
  • Constitutive ICAM-1 expression in K562 cells was not inhibited at the 25 ⁇ M level. The inhibition is selective in that oligonucleotide I inhibits induction of cell surface MHC-I by IFN- ⁇ , but does not inhibit induction by either IFN- ⁇ or IFN-3. ICAM-1 induction by TNF- ⁇ was also not inhibited.
  • the resultant sT2a successfully enhanced the oligonucleotide-specific blockage of IFN- ⁇ -mediated cell surface expression of the human MHC-II molecule, HLA-DR, which is a representative IFN- ⁇ inducible immune receptor as demonstrated in the examples section below.
  • RPE cells with sT2a can effectively thwart the recovery of IFN- ⁇ -mediated transcription and cell surface expression of HLA-DR for up to 15 days.
  • oligonucleotide (s) capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen is used herein to refer to oligonucleotides capable of inhibiting the IFN- ⁇ mediated induction of any surface antigen, preferably MHC-I, MHC- II and/or ICAM-1 induction. While these oligonucleotides may, under some circumstances, have regulatory effects beyond the IFN- ⁇ mediated induction transplantation antigen cell surface expression, they tend to have negligible effects on the constitutive expression of transplantation antigens, as well as negligible effects on the induction of transplantation antigens by IFN- ⁇ , IFN-jS, and TNF- ⁇ .
  • “Oligonucleotides capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen” may include a subset of oligonucleotides capable of binding to a portion of a transplantation antigen nucleotide sequence as described above. Alternately, this class of oligonucleotides may encompass a sub set of oligonucleotides that are not capable of binding to such sequences. The combined set of oligonucleotides tend to be less than 100 nucleotides in length, preferably between about 10 and about 50 nucleotides in length.
  • oligonucleotides all contain at least two guanine bases, preferably at least about 20% of bases are guanine, most preferably about 33% of bases are guanine.
  • Oligonucleotides capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen may be derived from guanine-rich regions of the MHC-II X-box, or generated from random sequences.
  • oligonucleotides may contain any of the chemical modifications in an oligonucleotide of the invention described above, including but not limited to modifications made to sugars, bases, and internucleotide linkages, as well as the inclusion of analogous forms of purines and pyrimidines and 5' and 3' organic capping groups.
  • At least one phosphodiester internucleotide linkage of the oligonucleotide is replaced by an internucleotide linkage selected from the group consisting of phosphorothioate and methylphosphonate. More preferably, a majority of the phosphodiester internucleotide linkages of the oligonucleotide are replaced by phosphorothioate groups. Most preferably, all of the phosphodiester internucleotide linkages are replaced by phosphorothioate internucleotide linkages.
  • These oligonucleotides are also preferably modified with a 3 'hydroxyalkylamine. Most preferably the 3 ' terminal OH of these oligonucleotides are substituted with a 3' -CH 2 CH (OH) CH 2 NH 2 group.
  • IFN- ⁇ -induced cell surface expression of a transplantation antigen may be derived from the following oligonucleotide sequences:
  • any of these sequences or fragments thereof may serve as a portion of the oligonucleotide or as the entire oligonucleotide.
  • the identity and relative position of the guanine bases are maintained, while the adenine, cytosine and thymine bases may be replaced by any non-guanine base. More preferably the identity and relative position of the guanine and adenine bases are maintained, while the cytosine and thymine bases may be replaced by any non-guanine base.
  • an "oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen” may be determined empirically by one of skill in the art, using any assay that can measure the cell surface expression of transplantation antigens before and after the administration of a dose of IFN- ⁇ .
  • These assays preferably utilize one or more labeled antibodies which specifically bind to a transplantation antigen and may include, for example, those assays disclosed in the Examples section below.
  • an "oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen" reduce the IFN- ⁇ -induced cell surface expression of at least one transplantation antigen by a minimum of at least about 25%, preferably by at least about 50%, more preferably by at least about 90%, most preferably by essentially 100%.
  • the oligonucleotides described above are used in a method of treatment to make a transplantation antigen-depleted cell from a normal target cell.
  • the cells are created by incubation of the cell with one or more of the above- described oligonucleotides under standard conditions for uptake of nucleic acids, including electroporation or lipofection.
  • the oligonucleotides can be modified or co-administered for targeted delivery to the nucleus.
  • the cell nucleus is a likely preferred site for action of the triplex-forming oligonucleotides of this invention, due to the location therein of the cellular transcription and replication machinery.
  • improved oligonucleotide stability is expected in the nucleus due to: (1) lower levels of DNases and RNases; (2) higher oligonucleotide concentrations due to lower total volume; (3) higher concentrations of key enzymes such as RNase H implicated in the mechanism of action of these oligonucleotides.
  • the cytoplasm is the likely preferred site for action of the traditional antisense oligonucleotides of this invention.
  • a primary path for nuclear transport is the nuclear pore.
  • Targeted delivery can thus be accomplished by derivatizing the oligonucleotides by attaching a nuclear localization signal.
  • a nuclear localization signal is the heptapeptide PKKKRKV (pro-lys-lys-lys-arg-lys-val) .
  • the target cell is selected from corneal endothelial cells, retinal pigmented epithelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells, hepatic cells, lymphoid cells, fibroblasts, epithelial cells, chondrocytes, endocrine cells, renal cells, cardiac muscle cells, endothelial cells, and hair follicle cells.
  • the target cell is selected from corneal endothelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells, epithelial cells, endothelial cells, and hepatic cells.
  • the above-described oligonucleotides may be incorporated into an expression vector through methods well known in the art, and then inserted into the target cell via standard techniques such as electroporation, lipofection, or calcium phosphate or calcium salt mediation.
  • the desired oligonucleotides are produced in situ by the expression vector, and the target cell will continue to express the oligonucleotides for at least a period of time following transplant.
  • this invention is applicable to the field of solid organ transplants.
  • Organs are normally perfused ex vivo prior to transplantation.
  • transplantation antigen-depleted cells can be created from perfusion-accessible cells in the organ to create a transplantation antigen-depleted organ useful in solid organ transplants.
  • the oligonucleotides of this invention are useful in creating the transplantation antigen-depleted cells of this invention. These cells are then directly transplanted to an individual . This technique can be used for any individual with an immune system, including humans.
  • the oligonucleotides of this invention are also useful in treating autoimmune diseases characterized by dysfunctional or aberrant expression of a transplantation antigen.
  • the oligonucleotides described herein may be administered in an amount sufficient to inhibit expression of the transplantation antigen.
  • the present invention encompasses methods for modulating the expression of a polypeptide, wherein the expression is regulated by IFN- ⁇ , said method comprising, exposing a cell to an oligonucleotide capable of inhibiting the IFN- ⁇ -induced cell surface expression of a transplantation antigen, said oligonucleotide being present in an amount sufficient to disrupt the IFN- ⁇ - regulated expression of said polypeptide in said cell.
  • These methods can be used to effect the level of expression of any polypeptide that is regulated by IFN- ⁇ , whether or not that polypeptide is a transplantation antigen. Further, these methods can be used to affect both IFN- ⁇ -induced and IFN- ⁇ -inhibited expression.
  • any oligonucleotide capable of inhibiting the IFN- ⁇ - induced cell surface expression of a transplantation antigen may be used ex vivo for administration to cells, tissues or organs, or in vivo for administration to an individual, preferably a human.
  • these methods use oligonucleotides selected from the group consisting of I 5' GGG GTT GGT TGT GTT GGG TGT TGT GT -RNH 2 ,
  • oligonucleotides of the invention interfere with or inhibit the production of one or more transplantation antigens is not always established, and is not a part of the invention.
  • the oligonucleotides of the invention are either characterized by their capability to bind to a specific target nucleotide sequence or their capability to inhibit the IFN- ⁇ -induced cell surface expression of a transplantation antigen, regardless of the mechanisms of binding, the mechanisms of IFN- ⁇ -induced protein expression, or the mechanism of the effect thereof.
  • Oligonucleotide action may be proposed for oligonucleotide action. Without wishing to be bound by any theory of action of the oligonucleotides according to the invention, several proposed mechanisms have been examined and an indication of how the data support or do not support the mechanism as a candidate has been made. Oligonucleotides are known to exert therapeutic effects on cells via several known mechanisms and are currently being investigated as therapeutic agents in a variety of strategies. These include the mRNA directed antisense, antigene directed triple helix, DNA binding protein directed DNA decoys, and aptamer strategies. Oligonucleotide treatment sometimes produces effects through unknown mechanisms and questions relating both to side effects and to "appropriate" controls have been discussed in the antisense literature.
  • the oligonucleotide directed to 3 ' untranslated region was found to inhibit ICAM-1 mRNA levels while the AUG codon targeted oligonucleotide did not and Chiang et al . suggested that the two oligonucleotides acted via distinct mechanisms.
  • Chiang et al . also found that IFN- ⁇ induced ICAM-1 was more sensitive to phosphorothioate oligo-mediated down regulation than either IL-1 ⁇ or TNF- ⁇ induced ICAM-1. This is may be due in part because the phosphorothioates used exhibit weak IFN- ⁇ inhibitory activity in addition to the other mechanisms that result in inhibition of IL-1 or TNF- ⁇ induced ICAM-1.
  • the MHC gene locus encodes a family of heterodimeric proteins that are involved in antigen presentation to T lymphocytes.
  • the protein ICAM-1 (CD54) belongs to the immunoglobulin superfamily, binds LFA-1 (CDlla/CD18) and mediates lymphocyte-target cell adhesion. These proteins and processes are central to the initiation of T cell-mediated responses and initiation of graft rejection.
  • the MHC genes and ICAM-1 are up-regulated by IFN- ⁇ , a powerful immunodulatory cytokine that is produced by lymphocytes and natural killer cells. IFN- ⁇ is known to induce or enhance expression of MHC-I, MHC-II and ICAM-1 proteins in a variety of cell types by transcriptional enhancement.
  • IFN- ⁇ also acts at the cellular level in the immune system, primes macrophages, and stimulates natural killer cells. These and other immunomodulatory effects of IFN- ⁇ increase the immunogenicity of tissues and enhance both antigen specific and nonspecific cell-mediated host response. However, these responses may be undesirable in those clinical situations where they promote graft rejection and inflammation.
  • Oligonucleotide I is apparently a representative of a family of oligodeoxynucleotides having specific and selective activities. Since a variety of cell surface markers are impacted by oligonucleotide I it may be suggested that an early step or steps in the regulation of genes by IFN- ⁇ is probably modulated. This is supported by the surprising similarities in the dose response curves for all three markers in K562 cells.
  • oligonucleotide I modulation of separate and late MHC-I and ICAM-1 unique steps by oligonucleotide I would likely have resulted in dose response curves differing significantly from each other, unless the changes were orchestrated coordinately on all the pathways involved.
  • An aptamer-like mechanism, in which oligonucleotides bind IFN- ⁇ , IFN- ⁇ receptor or other essential accessory proteins in the IFN- ⁇ induction process is plausible and not inconsistent with the initially observed data.
  • IFN- ⁇ has a pi of 8.6-8.7 and is positively charged at physiological pH. Further, the carboxyl terminus of IFN- ⁇ is essential for activity and has several positively charged amino acids that could possibly interact with negatively charged oligonucleotides via charge-charge interactions.
  • Such a pathway can plausibly inhibit the effects of IFN- ⁇ provided the structural requirements needed for efficient charge-charge interaction account for the differences in activity between oligonucleotides II and I.
  • the differences in activity may however be due to differences in half-lives between apparently active and inactive oligonucleotides caused by differences in nuclease susceptibility. It may also be contemplated that active oligonucleotides, through some undetermined mechanism, accelerate the rate at which IFN- ⁇ is committed to a futile degradative pathway or compartment.
  • IFN- ⁇ is essential for activity and that active oligonucleotides block this internalization process. All the mechanisms outlined so far act outside the cell or on the cell membrane. Oligonucleotides are taken up, albeit inefficiently and heterogeneously by cells and could enter cells and either bind to DNA binding proteins or alter binding of these proteins to DNA thereby inhibiting induction by IFN- ⁇ . However: i) the same IFN response element (IRE) is necessary for responsiveness to IFN- ⁇ , IFN-/S and IFN- ⁇ (Sugita et al . (1987) ; Korber et al .
  • IRE IFN response element
  • Oligonucleotides II and VI unlike oligonucleotide I appear to enhance IFN- ⁇ mediated MHC-I and ICAM-1 expression over the concentration range examined.
  • Several mechanisms including inhibition of nucleases, the displacement of mRNA stabilizing proteins and the modulation of the DNA binding proteins of genes, or aptomers could cause such enhancement.
  • oligonucleotides act at an early, membrane-associated step in the IFN- ⁇ signal transduction pathway.
  • the results shown in the Examples suggest that Oligonucleotide I may act at an early, membrane-associated step in the IFN- ⁇ signal transduction pathway.
  • An understanding is emerging of certain parts of the IFN- ⁇ transduction pathway, based upon studies in model systems. In such model systems, upon IFN- ⁇ binding to its receptor complex, two membrane-associated proteins, tyrosine kinases JAK1 and JAK2, are recruited to the receptor complex. This leads to simultaneous
  • oligonucleotides according to the invention as powerful agents for preventing allograft rejection, either as part of an ex vivo treatment to suppress expression of transplant antigens on grafts prior to transplantation, or in anti-rejection therapeutic agents.
  • sT2a The unique phosphorothioate and 3'hydroxyalkylamine double-modified oligonucleotide, sT2a, provides the novel property of long-term inhibition of IFN- ⁇ mediated transcription of cell surface expression of the human MHC-II molecular, HLA-DR. The evidence indicates that this long term inhibitory property of sT2a is associated with specific characteristics of intracellular accumulation, intracellular localization and resistance to nucleases.
  • Oligonucleotides and their analogs have been shown to specifically inhibit gene expression.
  • the potential success of oligonucleotides as therapeutic agents in the control of diseases and pathological processes at the genomic level rely on their ability to exert a potent and long term biological effect at a low dose.
  • significant inhibitory effects on gene expression were observed (Wickstrom, et al . , Proc. Natl. Acad. Sci. USA (1988) S _ :1028-1032; Zamecnik, et al . Proc. Natl. Acad. Sci. USA (1986) 11:4143-4146; and Harel-Bellan, et al . J.
  • oligonucleotide activity in cell culture has been demonstrated with phosphorothioates and in oligonucleotides modified at the 3' terminus. However, they are known to bind with lower affinities to double stranded DNA targets and single stranded DNA and RNA targets than do their DNA analogs (Stein, et al . Nucleic Acid Res. (1988) 16 . :3209-3221) . Assuming that binding affinity can be taken as a measure of molar potency, phosphorothioates have a lower molar potency than phosphodiester oligonucleotides. It is likely that this relationship extends to the 3'alkylamine analogs.
  • Phosphorothioates with a sulfur replacement at each internucleotide linkage, provide a nucleotide backbone that is more stable to nuclease attack and, which by many reports, is linked to their improved efficacy at lower doses (Reed, et al . Cancer Res . (1990) 5J):6565-6570; Casenave, et al . Nucleic Acid Res. (1989) 37:4255-4273; and Matsukura, et al . Proc. Natl. Acad. Sci. USA (1987) .84 . :7706-7710) .
  • the stability data alone is sufficient to explain the differences in duration of effect, but cannot and would not be expected to account for differences in acute activity.
  • the data herein suggest that differences in cellular association and intracellular distribution also alter the cellular availability of the oligonucleotide, and that these properties may contribute to both differences in acute activity and duration of effect.
  • 3'hydroxyalkylamine modified oligonucleotides are internalized (Orson, et al. (1991); Zhao, et al . Antisense Res. Dev. (1993) 3_:53-66; and Iversen, et al . Antisense Res. Dev. (1992) 2:211-222) .
  • Some studies suggest that these charged oligonucleotide analogs enter the cell via an endocytic process (Yakubov, et al . Proc. Natl. Acad. Sci. USA (1989) 8j[-6454-6458) and that transport across the cell membrane is mediated by a 80 kDa surface protein (Loke, et al . Proc. Natl. Acad.
  • sT2a oligonucleotide The prolonged effect of sT2a oligonucleotide clearly provides a promising new strategy for the control of the immunomodulatory effects specific for IFN- ⁇ .
  • the sT2a oligonucleotide may have therapeutic application in prevention of allograft rejection, blocking acute inflammation and treatment of certain autoimmune diseases. Exhibiting such an increased duration of biological effect in vi tro suggests that a phosphorothioate backbone-3'hydroxyalkylamine modification of an oligonucleotide could be an effective therapeutic tool in the prolonged inhibition of gene expression.
  • All cell lines were obtained from the Cell Culture Facility, University of California and were maintained at 37 °C with 5 % carbon dioxide in RPMI 1640 containing 10 % heat inactivated (56 °C for 30 minutes) fetal bovine serum (FBS) (GIBCO-BRL, Grand Island, NY) , 100 U/mL penicillin and 10 ⁇ g/mL streptomycin.
  • FBS fetal bovine serum
  • the human myelogenous leukemia derived K562 cell was selected as the primary model system because it has a very low background level of constitutive MHC-I that can be induced with IFN- ⁇ .
  • the Burkitts lymphoma derived Raji cell line, the uterine carcinoma derived HeLa S3 cell line, and the T cell lymphoma derived HUT78 were studied for comparison.
  • Recombinant human IFN- ⁇ (Collaborative Research Incorporated, Bedford, MA) was diluted in Dulbecco's PBS (D-PBS) to give 50 U/ ⁇ L, stored at -70 °C in aliquots, and thawed prior to use.
  • D-PBS Dulbecco's PBS
  • Recombinant human IFN- ⁇ (Chemicon, Temecula, CA) and IFN-? (Accurate Chemical Corporation, Westbury, NY) were diluted in D-PBS to 100 U/ ⁇ L and 1000 U/ ⁇ L respectively and stored at -20 °C. Aliquots of recombinant TNF- ⁇ (Genzyme Corporation, Cambridge, MA) were stored at -70 °C.
  • Oligonucleotides were synthesized using standard phosphoramidite protocols and purified by high performance liquid chromatography (Keystone Laboratories, Menlo Park, CA) . Oligonucleotide were stored at -20 °C as a 1250 ⁇ M stock solution in twice autoclaved diethyl pyrocarbonate treated deionized water.
  • Samples in triplicate were set-up in Falcon 2051 tubes and made up of 0.5 x 10 6 K562 cells and 0.25 ml of either: a) RPMI 1640; b) RPMI 1640 and 800 U/mL IFN- ⁇ ; or c) RPMI 1640, 800 U/mL IFN- ⁇ and oligonucleotide.
  • IFN- ⁇ and IFN-3 were used at 6400 U/mL in place of IFN- ⁇ .
  • RPMI 1640 containing heat inactivated (65 °C for 30 minutes) FBS was added to give a final FBS concentration of 10 % in a volume of 1 mL.
  • Samples were drawn from each tube at predetermined times and stained for flow cytometry using fluorescein isothiocyanate conjugated mouse mAb to human MHC-I heavy chain, ⁇ 2 microglobulin, ICAM-1 and a mouse IgG 2b control.
  • Fluorescein isothiocyanate conjugated mouse mAb to human MHC-I heavy chain, ⁇ 2 microglobulin and a mouse IgG 2b control were purchased from Olympus, Lake Success, NY.
  • the MHC-I heavy chain antibody recognizes an SDS stable determinant on the MHC-I heavy chain associated with ⁇ 2 microglobulin.
  • fluorescein isothiocyanate conjugated mouse monoclonals to human ICAM-1 and an IgG x control were purchased from AMAC (Westbrook, ME) .
  • Fluorescein isothiocyanate conjugated mouse monoclonals to human MHC-II DR and transferrin receptor were purchased from Becton Dickinson (San Jose, CA) .
  • Propidium iodide 50 ⁇ L of 50 mg/mL was added prior to cytometry to exclude dead cells from analysis.
  • the calibration curve used to determine the number of specific antibody binding sites on each sample was obtained by concomitantly staining 50 ⁇ L of Quantum Simply Cellular (Flow Cytometry Standards Corporation, Research Triangle, NC) beads with each antibody.
  • the data were analyzed using Lysys II software (Becton Dickinson, San Jose, CA) on a Hewlett Packard computer.
  • Fig. 1 summarizes data obtained with an antibody that recognizes a SDS stable determinant on the MHC-I heavy chain, and shows that oligonucleotide I inhibits IFN- ⁇ mediated induction of MHC-I heavy chain proteins on the cell surface at 24, 48 and 72 hours.
  • the human MHC-I protein is heterodimeric and consists of a 45 kD polymorphic heavy chain closely associated with ⁇ 2 microglobulin, a nonpolymorphic, 12 kD, light chain.
  • Figs. 2(a) through 2(c) shows that oligonucleotide I concomitantly inhibits the expression of ⁇ 2 microglobulin.
  • Figs. 3(a) through (c) shows that oligonucleotide I also inhibits induction of ICAM-1.
  • the data are normalized and expressed as a percentage of the protein expression observed with IFN treatment so as to allow a direct comparison of the results obtained with the various markers. From such a comparison, it is evident that the dose response relationships at any given time for all three markers are surprisingly similar in K562 cells.
  • oligonucleotide I inhibits an early step in the cascade of IFN- ⁇ mediated enhancement of cell surface MHC-I and ICAM-1 and the similarity in the dose response relationships for all three markers suggests that an early step or steps in the induction of proteins by IFN- ⁇ is probably altered by oligonucleotide I.
  • Constitutive ICAM-1 is unaffected because K562 cells treated with 25 ⁇ M of either oligonucleotide I or II in the absence of IFN- ⁇ did not decrease ICAM-1 expression (data not shown) .
  • Oligonucleotide II does not exhibit dose dependent activity against IFN- ⁇ enhanced MHC-I or ICAM-1 at 24 and 72 hours. However, an unusual dose dependent up regulation of IFN- ⁇ enhanced MHC-I and ICAM-1 is seen with II at 48 hours.
  • oligonucleotides I, III, IV, V and VI were compared.
  • K562 cells were treated with 25 ⁇ M of each oligonucleotide and analyzed for MHC-I, ⁇ 2 microglobulin and ICAM-1 expression at 48 hours.
  • Figs. 4(a) through (c) are broadly similar for all three markers.
  • oligonucleotide I-VI Alignment of oligonucleotide I-VI using a dynamic programming algorithm did not reveal significant sequence similarity.
  • the only structural characteristics separating the active oligonucleotides from the inactive oligonucleotides is the presence of guanosine bases in active oligonucleotides (e.g. I and III) and the presence of cytosine bases (absence of guanosine) in the inactive oligonucleotides (e.g. II and VI) .
  • the structure-activity relationships have not been identifiable based on an examination of the oligonucleotides for the presence of hairpins and inverted repeats which may result in the formation of secondary structures or facilitate the binding of DNA binding proteins. Searches of the primate database in GenBank with oligonucleotides I-VI as probes did not turn up targets different from those against which the oligonucleotides were originally directed.
  • MHC-I can also be induced by the cytokines IFN- ⁇ , IFN-/3 (Lindahl et al . (1976)) and TNF- ⁇ (Collins et al . (1986)) .
  • Fig. 5(b) shows that, at 48 hours, oligonucleotide I selectively inhibits IFN- ⁇ mediated MHC-I but does not inhibit MHC-I induction by 6400 U/mL of either IFN- ⁇ or IFN- / S.
  • the flow cytometry results with the anti-? 2 microglobulin mAb confirmed those obtained with the anti-MHC-I heavy chain antibody (data not shown) .
  • IFN- ⁇ and IFN-3 do not induce cell surface ICAM-1 in K562 cells.
  • cells treated with IFN-3 and 25 ⁇ M of oligonucleotide I appear to express slightly lower levels of MHC-I heavy chain, but recover almost completely by 48 hours.
  • the IFN doses were selected to give comparable enhancements of cell surface MHC-I.
  • the results show that 25 ⁇ M oligonucleotide I inhibits IFN- ⁇ induction of MHC-I, but has no significant effect on induction by IFN- ⁇ and IFN-/? under these conditions.
  • the effect of oligonucleotide I on the TNF- ⁇ mediated enhancement of ICAM-1 (Rothlein et al .
  • oligonucleotide I may be specific for IFN- ⁇ .
  • oligonucleotide I The studies of the activity of oligonucleotide I were extended to Burkitts lymphoma-derived Raji cells, to uterine carcinoma derived HeLa S3 cells, and to T cell lymphoma derived HUT78 cells. These cell lines are reasonable in vi tro models for B cells, epithelial and mature T cells respectively. Cells were treated with 25 ⁇ M oligonucleotide I according to the protocol used in the K562 cell experiments and stained with monoclonals for MHC-I heavy chain, ⁇ 2 microglobulin, MHC-II DR and transferrin receptor protein expression at 48 hours.
  • oligonucleotide I does not appear to down regulate any of the markers examined.
  • the poor induction of these markers by IFN- ⁇ , coupled with relatively high standard deviation for the measures for MHC-I and ⁇ 2 microglobulin expression did not allow an absolute determination of oligonucleotide I activity in this cell line. It is clear however, that oligonucleotide I does not significantly impact the constitutive levels of any of the markers examined. The relative stability of oligonucleotide I in the presence of these metabolically active cells was not determined.
  • the second panel in Fig. 7 shows that MHC-I levels are enhanced significantly in HeLa S3 by IFN- ⁇ , and that oligonucleotide I is only slightly effective in inhibiting this induction.
  • This low level of inhibition is a departure from the experience with K562 cells but is not due to low relative stability of I in HeLa S3 cells because ICAM-1 (unlike MHC-I) induction is inhibited by I.
  • the observed differences in the activity of oligonucleotide I between HeLa S3 cells and K562 cells could therefore be attributed to differences in IFN- ⁇ or oligonucleotide I sensitivities of the MHC-I or to other factors in the two cell lines.
  • oligonucleotide I is effective in inhibiting DR induction in HeLa S3, even though only a modest induction was observed at 48 hours. Unlike the other markers examined, the transferrin receptor is down regulated by IFN- ⁇ in the HeLa S3 cell line and 25 ⁇ M oligonucleotide I appears to effectively block this down regulation. Thus, oligonucleotide I is capable of inhibiting both the up regulation and the down regulation of IFN- ⁇ regulated genes.
  • oligonucleotide I In summary, the ability of oligonucleotide I to inhibit IFN- ⁇ induction of specific proteins is not restricted to one cell line, suggesting that oligonucleotide I and similarly acting oligonucleotides intervene directly in the cascade of events initiated by IFN- ⁇ . Finally, the low activity against MHC-I in HeLa S3 cells suggests that the enhancement of the same cell surface marker in different cell types may have different sensitivities/susceptibilities to inhibition by this drug class.
  • the human cervical carcinoma cell line, HeLa S3 was grown in RPMI 1640 containing 25 ⁇ M HEPES buffer and supplemented with 10% fetal calf serum (FCS) (Gibco, Gaithersburg, MD) , 100 U/mL penicillin, 100 U/mL streptomycin and 2 mM L-glutamine (Gibco, Gaithersburg, MD) .
  • FCS fetal calf serum
  • RPE cells were maintained in DME H16 (Gibco, Grand Island, NY) supplemented with 15% FCS (Sterile Systems, Inc., Logan, UT) , 300 ⁇ g/mL L-glutamine, 50 ⁇ g/mL fentamicin (Sigma, St. Louis, MO) , 100 U/mL penicillin and streptomycin and 2.5 ⁇ g/mL fungizone (Squibb, Princeton, NJ) as described in Song, et al. J. Cell Phvsiol. (1990) 141:196-203.
  • Basic fibroblast growth factor which was purified from bovine pituitary and brain (as described in Gospodarowicz J. Biol. Chem.
  • Viability of cells was determined by simultaneous staining with ethidium bromide and acridine orange or by co-staining cells intended for immunofluorescence analysis with 10 ⁇ g/mL propidium iodide (all from Sigma, St. Louis, MO) .
  • oligonucleotide I without the addition of the R group was identified as T2 and used herein along with the following control oligonucleotides :
  • T2 oligomer and control oligomers which include the following: modification with a 3' hydroxypropyl amine; modification with a phosphorothioate, methylphosphonate; or modification with phosphorothioate-3 'hydroxypropyl amine as listed in Table 4 below.
  • Modification I represents modification with the phosphorothioate-3 'hydroxypropyl
  • Modification II represents modification with the 3 'hydroxypropyl amine.
  • Modification III represents modification with phosphorothioate.
  • Modification IV represents modification with the methylphosphonate.
  • Modification V represents the unmodified oligonucleotide.
  • oligonucleotides revealed a purity of greater than 97% of the full-length modified oligonucleotide. All oligonucleotides were then reconstituted in sterile deionized water, adjusted to 400 ⁇ M, which was determined by evaluating the optical density at 260 nm (OD 260nm ) .
  • Oligonucleotides were 5 'end-labeled with 32 P ⁇ -ATP using T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN) by the exchange reaction method. Fluorescently-labeled oligonucleotides were constructed by a computer-controlled synthesizer incorporating the label as fluorescein phosphoramidite (Clontech, Palo Alto, CA) at the base positions 4 and 23. All fluorescently-labeled oligonucleotides were subsequently purified by HPLC and stored at -20°C and a 200 ⁇ M solution in the dark.
  • Incubations were performed in the dark at 4°C for 30 minutes using saturating mAb concentrations. Unincorporated label was removed by washing in PBS prior to analysis with a FACSort flow cytometer (Becton-Dickinson, San Jose, CA) .
  • Antigen density was indirectly determined in gated live cells and expressed as the mean channel of fluorescence (MCF) .
  • MCF mean channel of fluorescence
  • Surface expression of HLA-DR was represented as the mean channel shift (MCS) obtained by subtracting the MCF of FITC-anti IgG 2a -stained cells from the MCF of FITC-anti HLA DR stained cells.
  • HeLa cells (4-6 x 10 6 ) from each test preparation were homogenized in lysis buffer (4 M guanidinium isothiocyanate (Gibco BRL, Gaithersburg, MD) ) containing 0.1 M Tris, pH 7.5, 0.01 M EDTA, pH 8, 1% ⁇ - mercaptoethanol and 10% N-laurylsarcosine (all from Sigma, St. Louis, MO) followed by isolation of total cellular RNA using the method previously described by Chomczynski and Sacchi Anal. Biochem. (1987) 162 :156-159. Estimates of RNA concentration were evaluated following OD 260nm determination.
  • RNA samples were performed using an RNase protection assay kit (Ambion, Austin, TX) .
  • 32 P- riboprobes were generated using a Maxiscript in vi tro transcription kit (Ambion, Austin, TX) and ⁇ 32 P-UTP (10 mCi/mL, NEN DuPont, Boston, MA) .
  • Haelll-digested X174 (Gibco BRL, Gaithersburg, MD) was 5' end-labeled with 32 P- ⁇ ATP (NEN DuPont) using T4 polynucleotide kinase (Boehringer-Mannheim) and served as molecular weight markers in RNase protection assays.
  • Isolated RNA (20 ⁇ g/lane) was hybridized to a T3 RNA polymerase generated 32 P-RNA probe (3-5 x 10 5 cpm) antisense to a 1.2 kb fragment from the human DR alpha gene.
  • the DR alpha fragment was contained within plasmid DR A pBS M13 and lineariz-ed with EcoRI prior to use.
  • the remaining probe which was protected from RNase A and Tl digestion (at 37 °C for 30 minutes) was recovered by phenol/chloroform extraction and precipitated. Samples were heated for 5 minutes at 95° C prior to electrophoresis for 2 hours at 250V on a 6% denaturing polyacrylamide gel.
  • T2a oligonucleotide was studied using 12 replicate samples of 2 x 10 5 HeLa cells per test. The cells were treated with 4 ⁇ M sT2a or T2a and IFN- ⁇ as described above. On Days 4, 8, 10 and 12, aliquots of 0.5 mL (approximately 4 x 10 B cells) from each tube were washed and recultured in 2 mL oligonucleotide-free culture medium containing 200 U/mL IFN- ⁇ . A second aliquot of cells from 4 replicates of each sample was prepared for immunofluorescence analysis and the remainder of the cells were pooled and prepared for RNA analysis. Immunofluorescence analysis alone was performed on Day 15.
  • RPE cells grown to 70-80% confluence in 4-well cell culture chamber slides were washed twice with PBS and treated with 20 ⁇ M fluorescence sT2a or T2a oligonucleotide in a serum-free culture media without phenol red for 5 and 120 minutes at 37°.C and 5% C0 2 .
  • Cells were again washed twice with PBS, fixed with 3.7% paraformaldehyde, and viewed with confocal microscopy within 6 hours of slide preparation. Control cell populations were treated similarly with equal volumes of either serum-free media or 20 ⁇ M free fluorescein in serum-free media.
  • Microscopy was performed on a Biorad (Cambridge, MA) MRC600 confocal laser scanning microscopy system equipped with an Argon ion laser exciting maximally at 488 nm and 514 nm and operating under CoMoS software.
  • Neutral density (ND) filters were set to utilize 1% laser power and linearity was maintained between input photons and output voltage.
  • Fig. 9(a) shows that in HeLa cells sT2a and T2a (see Table 4 for nomenclature of modification of T2 and control oligonucleotides) were similarly effective in blocking IFN- ⁇ -induction of MHC-II DR expression, both showing greater activity than sT2 over the dose range 0.5 - 4 ⁇ M. It is noteworthy that 3 'modification significantly augmented the inhibitory activity of a phosphorothioate-modified oligonucleotide sT2. The double-modified sT2a was also more effective than either the unmodified, umT2 or the methylphosphonate, mpT2 oligonucleotides in the dose range 2 - 6 ⁇ M (Fig.
  • Fig. 9(b) illustrates the recovery of IFN- ⁇ -induced HLA-DR expression in sT2a and T2a-treated HeLa cells on Days 4 and 8. On subsequent test days, DR expression was recovering.
  • sT2a-mediated inhibition had only recovered to 75% of HLA-DR levels in cells treated with IFN- ⁇ alone.
  • a complete inhibition of the IFN- ⁇ -induced HLA-DR expression in T2a-treated cells was observed only by Day 4 but by Day 8, HLA-DR expression had recovered and paralleled that found in cells treated with IFN- ⁇ alone.
  • Fig. 11 shows a parallel study documenting the temporal effects of sT2a and T2a and T2a on HLA-DR ⁇ mRNA levels in HeLa cells. Treatment with sT2a completely inhibited transcription of DR ⁇ mRNA on Days 4 (lane 5) and 8 (lane 7) .
  • Fig. 12 shows results from experiments which compared the ineffectiveness of the control oligonucleotides (listed in Table 4) in inhibiting induced HLA-DR expression.
  • Fig. 12(a) shows that there is no apparent dose effect for either COla or sCOla between 1 - 4 ⁇ M although the date does suggest a similar modest non-specific effect for both.
  • treatment COla Fig. 11, lane 13
  • sCOla Fig. 11, lane 14
  • Controls sC02a, C02a and C03a were similarly incapable of producing a dose effect in the 2 - 4 ⁇ M range.
  • Fig. 12(b) shows a within study comparison of the controls at 4 ⁇ M each. There was no significant difference.
  • oligonucleotide treatment alone gave no apparent additional cytotoxicity and any changes in viability were associated only with IFN- ⁇ treatment
  • the patterns of degradation are shown in Fig. 13.
  • the principal degradation products of T2a and umT2 were single labeled fragments released by 5 ' exonuclease or highly processive 3 ' exonuclease activity, and free 32 P-inorganic phosphate released by phosphate activity.
  • Less apparent degradation of full length T2a suggests modification with 3'hydroxyalkylamine provided protection from 3 ' exonucleases.
  • the absence of released 5 ' terminal 32 P-nucleotide fragments from sT2 and sT2a suggests they were resistant to 5' exonucleases.
  • the degradation profile of sT2a revealed a low occurrence of 32 P-nucleotide fragments and free 32 P-inorganic phosphate, suggesting that the combination of phosphorothioate backbone with 3'hydroxyalkylamine thwarted both exonuclease and phosphatase activity.
  • Fig. 14 shows that sT2a exhibited the least degradation with 65% remaining intact in extracellular supernatants (S, panel A) and cell lysates (L, panel B) after 72 hours. In contrast, the percentage of full length sT2a remaining intact after a 72 hour incubation was 31% (S) and 23% (L) ,- the corresponding percentages for sT2a were 28% (S) and 29% (L) and for umT2 they were 10% (S) and 18% (L) . In RPE cells, degradation of sT2a and T2a was more severe than in HeLa cells (Fig. 15) . However, sT2a was still the most stable. In contrast, less than 15% T2a remained intact after 24 hours.
  • Fig. 17 illustrates the comparative uptake and localization of properties of the two oligonucleotides after 5 and 120 minutes of cell incubation. A significant and comparable cell surface accumulation of both oligonucleotides was observed within 5 minutes of incubation. At 120 minutes, the fluorescence distribution arising from the T2a and sT2a oligonucleotides follows diverging paths. Internalized T2a remained in punctate vesicles which demonstrated a progression from a primarily radial to a primarily perinuclear distribution (Fig. 17(a) , 17(c)) .
  • sT2a was also internalized as punctate vesicles but then demonstrated a time-dependent partitioning out of the vesicle and into the intracytoplasmic and intranuclear compartments (Fig. 17(b) , 17(d)) .
  • Fig. 17(b) , 17(d) intracytoplasmic and intranuclear compartments
  • oligodeoxynucleotides have been identified and evaluated that inhibit the effects of IFN- ⁇ in a variety of cell lines at micromolar concentrations, with emphasis on the inhibitory effects on the IFN- ⁇ mediated enhancement of two markers MHC-I and ICAM-1.
  • human fibroblasts (BUD- 8, ATCC) were grown in Eagle's MEM/Hank's BSS supplemented with non-essential amino acids, penicillin- streptomycin, and 10 % fetal calf serum.
  • IFN- ⁇ stimulation was done with 200 U/cc IFN- ⁇ (Collaborative Research) mixed in growth media.
  • Oligonucleotide I or control oligonucleotide Cl were added to some cells to a final concentration of 6.25 ⁇ M. After appropriate stimulation, cells were washed with cold PBS with 0.5 mM Na 3 V0 4 .
  • Cells were lysed with 50 mM Tris (pH 8.0), 300 mM NaCl, 0.5% NP-40, 10% glycerol, 0.2 mM EDTA, 2 mM EGTA, 1 mM DTT, 0.5 mM Na 3 V0 4 , and lx protease inhibitors (Pefabloc/leupeptin/ aprotinin) ; nuclear proteins were extracted for 30 minutes at 4 °C. Cell lysates were spun at 12,000 rpm for 15 mins, and supernatants were collected.
  • blots were probed with anti-phosphotyrosine mAbs (RC20H, Transduction Laboratories) or anti-stat91 mAbs (Transduction Laboratories) and visualized with the ECL system (Amersham) .
  • Electrophoresis mobility shift assays were optimized using an EMSA optimizing kit (Pharmacia) .
  • 125 i- IFN- ⁇ (Amersham) were allowed to bind with Oligonucleotide I (1 to 20 ⁇ M) at physiologic salt conditions, at room temperature for 30 mins.
  • Samples were resolved in 8 % polyacrylamide gels in Tris-glycine buffer (pH 8.6) and visualized with autoradiography for 48-72 hours.
  • Oligonucleotide I Blocks Phosphorylation of stat91 Human fibroblasts (BUD-8) were stimulated with 200 U/cc IFN- ⁇ in the presence or absence of 6.25 ⁇ M Oligonucleotide I with anti-stat91 mAbs, proteins were extracted from the cells, and immunoprecipitated, followed by immunoblotting with anti-phosphotyrosine mAbs.
  • Fig. 18 shows the result of concurrent treatment of cells with IFN- ⁇ and Oligonucleotide I or control
  • Oligonucleotide Cl Cells were treated with media alone (lanes 1, 2) ; with 200 U/cc IFN- ⁇ (lanes 3, 4) ; with IFN- ⁇ plus 6.25 ⁇ M Oligonucleotide I (lanes 5, 6) ; or with IFN- ⁇ plus 6.25 ⁇ M Oligonucleotide Cl (lanes 7, 8) for 15 mins at 37 °C, then lysed. Cellular proteins were extracted and immunoblotted as described above. The lower panel shows a 91 kD band present in all lanes, demonstrating that stat91 was successfully immunoprecipitated. When probed with anti-phosphotyrosine mAbs (upper panel) , only the lanes containing IFN- ⁇ alone or containing IFN- ⁇ plus Oligonucleotide Cl showed phosphorylation of stat91.
  • Fig. 19 shows the result of sequential treatment of cells with Oligonucleotide I and with IFN- ⁇ , separated by a washing step. Cells were first pretreated with 6.25 ⁇ M Oligonucleotide I (lanes 5, 6) or control Oligonucleotide Cl (lanes 7, 8) for 15 mins at 37 °C.
  • FIG. 20 shows the results of EMSA of 125 I-IFN- ⁇ with Oligonucleotide I.
  • Lane 1 contains molecular markers; lane 2 contains 125 I-IFN- ⁇ alone; lanes 3-7 contain IFN- ⁇ plus Oligonucleotide I at: 1 ⁇ M (lane 3), 3 ⁇ M (lane 4), 5 ⁇ M (lane 5), 10 ⁇ M (lane 6), and 20 ⁇ M (lane 7) ; lanes 8-10 contain 125 I-IFN- ⁇ plus 20 ⁇ M
  • Oligonucleotide I and: 200 U/cc IFN- ⁇ (lane 8) , 200 U/cc IFN-/3 (lane 9) , and 200 U/cc TNF- ⁇ (lane 10) .
  • IU 5-iodouracil-substituted oligonucleotides
  • IU substitution of Oligonuc2.eotide I at positions near each end and near the middle are employed.
  • ⁇ such IU-substituted Oligonucleotide I had the same effect as unsubstituted Oligonucleotide I, as reflected by degree of inhibition of surface HLA class I and ICAM expression upon IFN- ⁇ stimulation by K562 cells at 24 and 48 hours.
  • IU-substituted Oligonucleotide I is allowed to bind its target for 15 mins, followed by in si tu induction of crosslinking by exposing Oligonucleotide I-bound cells to UV at 311 nm for 30 mins. The cells are then lysed, and the membrane proteins are extracted and analyzed by SDS- PAGE. 5' -labelling of Oligonucleotide I with 32 P provides for identification of Oligonucleotide
  • radiolabeled (3- [ 125 I] iodotyrosyl) IFN- ⁇ was reconstituted in 20 ⁇ l water according to the manufacturers instructions, aliquoted and stored at -20°C.
  • oligo concentrations were obtained from spectrophotometric absorbance measurements at 260 nm by using a conversion factor of 33 ⁇ g oligo per unit of OD.
  • Protocol A Samples were prepared in triplicate. Each sample tube contained 0.5 x 10 6 K562 cells and 0.25 ml of either a) serum-free media b) serum-free media and 800 Units/ml IFN- ⁇ or c) serum-free media, 800 Units/ml IFN- ⁇ and oligo. For the specificity experiments, IFN- ⁇ was replaced by IFN- ⁇ (6400 U/ml) , IFN-? (6400 U/ml) , or TNF- ⁇ (800 U/ml) . These cytokine doses were selected to give cell surface MHC Class I enhancements comparable to 800 U/ml IFN- ⁇ .
  • Protocol B Protocol A was modified to eliminate the 1 hour preincubation of cells in serum-free medium.
  • the volume of medium was 1 ml and contained 90.5 x 10 6 K562 cells, 10% (65°C, 30 minutes) heat inactivated FBS, and the indicated concentrations of cytokines and/or oligo.
  • IgG 2 b control (Olympus, Lake Success, NY) were used.
  • the MHC Class I heavy chain antibody recognizes an SDS stable determinant on the MHC Class I heavy chain associated with ⁇ 2 microglobulin antibody recognizes both soluble and MHC Class I heavy chain associated ⁇ 2 microglobulin.
  • fluorescein isothiocyanate conjugated mouse monoclonals to human ICAM-1 (AMAC, Westbrook, ME) , MHC Class II DR (Becton Dickinson, San Jose, CA) , transferrin receptor (Becton Dickinson, San Jose, CA) and an IgGi control (AMAC, Westbrook, ME) .
  • the calibration curve used to determine the number of specific antibody binding sites on each sample was obtained by concomitantly staining 50 ⁇ l of Quantitative Simply Cellular (Flow Cytometry Standards Corporation, Research Triangle, NC) beads with each antibody.
  • Quantitative Simply Cellular Flow Cytometry Standards Corporation, Research Triangle, NC
  • This product contains 5 different populations of beads each with predetermined number of mouse antibody binding sites and allows a calibration curve of the mean number of antibody binding sites vs the mean channel fluorescence to be obtained.
  • the mean number of binding sites within each sample was computed from the mean channel fluorescence value by interpolation from the calibration curve.
  • the data at each time point were expressed as a percentage of the corresponding IFN- ⁇ induced value. This measure of the cell surface marker expression is independent of the antibody-antigen binding stoichiometry.
  • K562 cells were treated with 200 U/ml IFN- ⁇ according to Protocol B. At 0, 2, 4, 8, 12 and 24 hours, I was added to one set of cells to give a final concentration of 6.25 ⁇ M. Additional sets of cells were washed twice with 5 ml of serum-free media at 0, 2, 4, 8, 12 or 24 hours to remove external IFN- ⁇ and served as controls . Aliquots were drawn from each tube at 24 and 48 hours for flow cytometry.
  • K562 cells were treated with 6.25 ⁇ M I for 0, 2, 4, 8, or 12 hours according to Protocol B. The cells were washed twice to remove externally accessible I and 200 U/ml IFN- ⁇ was added. Aliquots were analyzed for ICAM-1 expression by flow cytometry 12 and 36 hours after IFN- ⁇ addition.
  • Binding studies were carried out to evaluate the effect of I and II on the cell surface binding of IFN- ⁇ spiked with 125 I labeled IFN- ⁇ .
  • each tube contained 2 x 10 6 K562 cells treated with labeled IFN- ⁇ .
  • a series of labeled IFN- ⁇ concentrations was prepared by 1:2 serial dilution so as to give final IFN- ⁇ levels ranging from 5000 units/ml to 19 units/ml. Aliquots of the labeled IFN- ⁇ were analyzed in a Cobra AutoGamma (Downers Grove, IL) gamma counter and the specific activity calculated.
  • I or II was added to the experimental tubes to a final concentration of 10 ⁇ M.
  • the tubes were incubated on ice for 1 hour and then centrifuged to pellet the cells. Aliquots of the supernatant were removed to determine the free IFN- ⁇ concentration.
  • the cell pellet was washed three times with cold PBS containing 2% 65°C heat treated FBS and the radioactivity was quantitated in the gamma counter.
  • One set of experiments was carried out with a 100 fold excess of cold IFN- ⁇ to ensure that the radiolabeled IFN- ⁇ competed with cold IFN- ⁇ for cell surface binding, and to provide assurance that the behavior of the labeled cytokine did not differ significantly from that of the unlabeled material.
  • MHC Class I is synergistically induced by mixtures containing IFN- ⁇ and TNF- ⁇ .
  • IFN- ⁇ and TNF- ⁇ we evaluated the effect of 0 or 6.25 ⁇ M oligo I on K562 cells treated with 0, 5, 50, or 500 U/ml TNF- ⁇ with or without 50 or 200 U/ml IFN- ⁇ .
  • the 24-hour data for ⁇ 2 microglobulin and ICAM-l are summarized in Figure 26. Over a 100 fold range of TNF- ⁇ , in the absence of IFN- ⁇ , the addition of 7.125 ⁇ M I had no effect on the enhancement of IC7AM-1 expression, confirming that it is selective.
  • TNF- ⁇ alone does not significantly induce ⁇ 2 microglobulin levels, but mixtures of TNF- ⁇ and.50 U/ml IFN- ⁇ result in synergistic increases.
  • I is added to mixtures containing TNF- ⁇ and 50 U/ml IFN- ⁇ both the IFN- ⁇ mediated indication and the synergy are inhibited and expression returned to levels comparable to background.
  • the synergy observed with mixtures containing TNF- ⁇ and 200 U/ml IFN- ⁇ was higher than that observed with mixtures of TNF- ⁇ and 50 U/ml IFN- ⁇ . Addition of I was efficacious in inhibiting both the induction and synergy mediated by the IFN- ⁇ .
  • ICAM-l levels observed on the addition of I to mixtures of TNF- ⁇ and 50 U/ml IFN- ⁇ are always higher than those observed on the addition of I to TNF- ⁇ alone. This result suggests that only the IFN- ⁇ contribution is inhibited, and that the TNF- ⁇ mediated enhancement of ICAM-l is independent of I.
  • Raji cells constitutively express high levels of MHC Class I, MHC Class II DR and transferrin receptor. IFN- ⁇ did not significantly change these levels, and I does not appear to down regulate any of the markers examined (data not shown) .
  • HUT78 cells the IFN- ⁇ induced expression of ICAM-l was inhibited by I.
  • I can inhibit the upregulation of proteins by IFN- ⁇ , but does not alter constitutive levels of cell surface proteins.
  • IFN- ⁇ induces MHC Class I heavy chain, ICAM-l and MHC Class II DR in HeLa Se ( Figure 27) .
  • Oligo I inhibits MHC Class II DR and ICAM-l induction but in contrast to K562 cells, I has little or no effect on MHC Class I (or ⁇ 2 microglobulin, data not shown) expression.
  • the transferrin receptor is down regulated by IFN- ⁇ in HeLa S3 cells and 25 ⁇ M I also inhibits this down regulation.
  • I is capable of inhibiting both the up regulation and the down regulation of some IFN- ⁇ regulated genes.
  • This preincubation experiment was designed to determine whether the I remaining in cells after washing was sufficient to provide inhibitory activity, and to determine whether the site of action was accessible for the removal of I by washing the cells.
  • the data in Figure 29 show that washing abrogates the inhibitory activity of I.

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Abstract

L'invention concerne des cellules appauvries en antigènes de transplantation et des procédés de préparation des dites cellules à partir d'une cellule cible. On prépare la cellule appauvrie en antigènes de transplantation par exposition d'une cellule cible à un oligonucléotide pouvant soit se fixer à une séquence de nucléotides d'antigènes de transplantation, soit inhiber l'expression de la surface de la cellule provoquée par IFN-η d'un antigène de transplantation. Les oligonucléotides présentant une capacité de fixation à une séquence de nucléotides d'antigènes de transplantation possèdent chacun au moins un groupe de liaison internucléotide de phosphorothioate et, de préférence, une modification d'hydroxyalkylamine. L'invention concerne également des organes donneurs universels, des procédés de traitement d'individus atteints par une maladie auto-immune caractérisée par l'expression dysfonctionnelle d'un antigène de transplantation, ainsi que des oligonucléotides efficaces dans la mise en application de l'invention.
PCT/US1995/001198 1994-01-28 1995-01-26 Procede de preparation de cellules donneuses universelles WO1995020317A1 (fr)

Priority Applications (1)

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AU16956/95A AU1695695A (en) 1994-01-28 1995-01-26 Method for making universal donor cells

Applications Claiming Priority (6)

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US18843594A 1994-01-28 1994-01-28
US08/188,435 1994-01-28
US20613194A 1994-03-04 1994-03-04
US08/206,131 1994-03-04
US30546794A 1994-09-12 1994-09-12
US08/305,467 1994-09-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220195460A1 (en) * 2014-03-26 2022-06-23 Denovo Biopharma Llc Retroviral vector having immune-stimulating activity

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EUROPEAN JOURNAL OF IMMUNOLOGY, Volume 23, issued 1993, H. TANG et al., "The Effects of a Monoclonal Antibody to Interferon-gamma on Experimental Autoimmune Thyroiditis (EAT): Prevention of Disease and Decrease of EAT-Specific T Cells", pages 275-278. *
JOURNAL OF IMMUNOLOGY, Volume 151, No. 2, issued 15 July 1993, K.J. WIEDER et al., "Rapamycin Treatment Depresses Intragraft Expression of KC/MIP-2, Granzyme B and IFN-gamma in Rat Recipients of Cardiac Allografts", pages 1158-1166. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220195460A1 (en) * 2014-03-26 2022-06-23 Denovo Biopharma Llc Retroviral vector having immune-stimulating activity

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