CA2126692A1 - Method for making universal donor cells - Google Patents

Abstract

Transplantation antigen-depleted cells and methods for making such cells from a target cell are provided. The transplanta-tion antigen-depleted cell is made by exposing a target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence. The oligonucleotide is capable of binding to the nucleotide sequence according to Watson-Crick or triplex binding rules. Preferred transplantation antigens are the MHC class I or II antigens. Oligonucleotides useful in practicing this method are also provided.

Description

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WO93/14769 P~T/US93/00797 MET~OD FOR MAKING UNIVERSAL DONOR CELLS :~
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Technical Field This invention is related to therapeutics, transplantation and immunology. More specifically, it relates to a method for making cells that are more easily transplanted into a recipient host using oligonucleotides- 20 that interact with genes and gene products relating to transplantatio~ antigens expressed on the cell surace of transplanted cells.

Backqround Art _ -Anti-gene code molecules are short RNA or DNA
trans~ripts that are '`antisen~e" (i.e., complementary to a DNA or RNA strand in a Watson-Crick pairing manner) to - -a portion of the normal mRNA and are not translated.
Regulation of expre~sion of genes by anti-gene code RNA, one of the natural modes of gene regulation, was first recognized in prokaryotes~ Green, P.M. et al., Ann~ Rev. ~ ~
Biochem (lg86) 55:569. Natural anti-gene codes and artificial anti-gene codes have been used in p~okary~tes ~
to downrequlate prokaryotic proteins. Simmons, R.W. et -~~=- -al., Cell ~1983) 34:683; Mizuno, T. et al., Proc. Natl.
Acad. Sci. ~1984) 81:1966; Okamoto, X. et al., Proc.
Natl. P.cad. Sci. (1986) 83:5000; Pestka, S. et al., Proc.

W093/14769 ~ J-~j`f PCT/US93/00797 Natl. Acad. Sci. (1984) ~1:7525; Coleman, J. e;t al., Cell (1984) 37:429; Farnham, P.J. et a~., Proc. Nat~l. Acad. -~
Sci. (1985) 82:3978; Kindy, M.S. et al., Mol. Cell. Biol.
(1987) 7:2857.
S Artificial anti-gene cod-æ have also been synthesized and used to regulate eukaryotic gene expression~ Microinjection or transfection of t~ymidine ~kinase~(TK) anti-gene~ codes has beén~shown to;inhibit express~lon of the~ TX protein. Izant, J.G. et-al., Cell ~10 ~ 19~84~ 36:~1007; Kim,~S.K. ~et~al.,~Ce;~1 (1985)~42:129. ~;
Add~itlonally,~ short anti-gene CoAec~to the 5';
uneranslated~reg;ion of the thymidine kinase~ qene surcnssfully~downre~ulates protein~expression.~ Izant, J.G. et al.,~Science~(1985~)~229:345. Other examples of ~;~15 the regulation of eukaryotic gene express~ion by anti-gene cotes;are~the~pp6~6~c-src~g-ne~ by~LL~al~ fected full length gène~codes)~, and the~c-;fos~gène~ (by~ ~an :ant i-~ene code~sp ~ t ~5~' untranslate~ gi of the first exon~ Amini,~S.~et~al.,~Mo~ Cell.~iol.~(1986) :2305~;

2~0 ~ Holt,;~J.~T.~et~al.~ ~ c. a 1.~ ad~ Sci. (19~6)~8 :4794.
~é anti ~r.~c ~r-~e~have-be ~int constitutive-o~r~heterolo~ous~ inducible~promoters.
Synthet~ic;ollgomers~have also beèn~used to townreg~latè~ e cx ~ e~ on~of~ in~ ;lo 25~ leukemia~Gells,~ a*d~ ~ ~ y ~ ~ ickstrom, E.L. et ~ ~
al.,~Proc.-~Natl~ Acad~ Sc~ (198~8)~ 102~8, H~eikkila, R. ~`
et~al~ Nature~ 1987~ ~ 5445.~ C-m ~-an-~i~7ene code o ~ leotid-s havé~ n~`~ own~-to~ hLt;p oliferation n normal~hematopoiétic~cells. -Gewirtz,~A.M.~ et al., 30~ : sGience~ i(l988)~;242:1303.~; An anti-gene code`to a CD8 fragment~downr~ -ted tbe~expression of CD8~ mo}ecules on the~surfa¢e Or~ ~ n~¢ytotoxic T-cells. ~Hambor, J.E. et al., Proc.~ Natl. A~d.~Sci. ~1988)~ 4010. Lotteau et al.,~J.~ FY~.~ Med.~ 1989);169:~351~used ~eplsomàl Yectors to introducé~DR~A~coding~seguences~n 8-lymphoblastoid cell ~, ~ - , . .

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lines and downregulated the expression of DR A-DQ B mixed isotype heterodimers, but did not observe any chan~es in ~-the levels of iso~ype matched DR A DQ B heterodimers.
Anti-gene code oli~onucleotides may act to prevent transcription by inhibiting DNA or RNA
polymerase, by binding to m~NA and preventing ribosomal~,~
translation, ~y decreasing the stability of mRNA through ,' enhancement of mRNA degradation by RNase H, or by preventing or inhibiting the processing to mat~re mRNA.
Maher, L.J. et al., Science (1989) 245:725; Moser, H.E.
et al., Science (1987) 238:645; Melton, D.A. et al., Proc. Natl. Acad. Sci. (1985) 82:144; Gewirtz, A.M. et al. ! Science (1989) 245:180; Walder, R.Y. et al., Proc.
Natl. Acad. Sci. (1988) 85:5011. Absolute homoloqy between the target and the antisense sequences is preferred but not required for the inhibition. Holt, J.T., suDra.
Anti-gene code oligonucleotides may also form a triplex DNA structure with the intact duplex gene.
Moffat,- ~.S., Science (1991) ,252:1374-1375. This ,~
technique of making anti-gene code oligonucleotides involves the formation of a triplex structure according to certain binding rules. When this triplex structure is formed, in the promoter region of a gene, it has been sh~wn to disrupt transcription of that gene. Orson, F.M.
et al., Nuc. Acids Res. (1991) 19:3435-3441.
one set o~ genes potentially subject to regulation by anti-gene codes is the Human Leukocyte Antigen (HLA) complex, located on the short arm of chromosome 6. The HLA antigens are divided into two classes depending on their structure. The genetic loci denoted HLA-A -B, and -C code for the HLA Class I ' antigens, and HLA-DP, -DQ and -DR code for the HLA Class II antigens. ~ ~~-WO~3/14769 . ,~ PCT/US93/00797 ~ v~ ,~ ~4~

HLA Class II molecules are composed of two non-~ovalently linked glycoproteins, the ~ chain and the highly polymorphic ~ chain. Each chain contains one extracellular domain, a transmembrane segment and a 5 cytoplasmic tail. The structure of the ~ and ~ chains ;-and their genes have been elucidated. All knawn Class II
genes are similar in structure and encoded by exons l -

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 lS the gene has not been reported in the literature.
Regulation of HLA class II antigen expression occurs in part through a series of promoter regions such as the J, W, X (including X~ and X2), and Y boxes, and the gamma interferon response element. The X ~including X~
and X2 ) 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) (l99l) 88:
4304-4308.
HLA antigens are lmplicated 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 onIy 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. Druys are commonly used to control immune responses and prevent graft rejection, and death i5 often an indirect result of this drug administration.

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W093~14769 PCT/US93/00797 The drugs used to control immune responses -- usually cause a non-specific depression of the immune system. A patient with a depressed immune system is far more susceptible to develop 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.
It has been suggested that 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) 252:1700-1702, 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" r~duced in one or more HLA ¦~
antigens. The absence of certain HLA antigens on the surface of donor cells, tissues or organs comprising 2~ 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 ~ 25 immune system. Thus, the problem of rejection is - eliminated without nonspecific suppression of the immune - -~ystëm,~ and the immune system remains active to defend ag-ainst~infection and neoplasia.
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SummarY of the Invention ~ - A superior method of providing transplantation antigen-depleted/reduced cells has now been found. In - _~
---~--~accord with this invention, oligonucleotides that reduce _ the antigenicity of cells are designed to be able to bind in some fashion to a nucleotide sequence relating to a WO93~14769 ~ PCT/US93/00797 ~ _~.j ?~ 6-transplantation antigen, and prevent the expression of that antigen. Cells treated with these oligonucleotides will express significantly less of the targeted antigen, and when transplanted will be more easily tolerated by the recipient host. Although these oligonucleotides are designed to ~e capable of ~inding 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 cells may also be useful in 15 treating patients with autoimmune diseases. ;~
-In one aspect, this invention provides a method 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 or produced locally in an amount sufficient to make the target cell a transplantation antigen-depleted ~ --cell. The oligonucleotide is capable of binding to the 2S nucleotide sequence according to Watson-Crick or triplex - binding rules (which includes Hoogsteen-like bonds). In ~-preferred embodiments the transplantation antigen is an -MHC class I or II antigen.
In another-aspect of this invention, a transplantation antigen-depleted cell is provided, prepared by obtaining a target cell, and exposing the target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide se~uence, wherein the `
oligonucleotide is presented or produced locally in an W 0 93/14769 7 ` ~ ^` P ~ /US93/00~97 amount sufficient to make the target cell a transplantation antigen-depleted cell.
~ In yet another aspect of this invention, an oligonucleotide capable of binding to a double-stranded transplantation antigen nucleotide sequence is provided.
In still ano~her aspect of this invention, a universal donor organ is pro~ided, 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 i5 presented or produced locally in an amount sufficient to make the target organ a universal donor organ.
- In a further aspect of this invention, a method of treating an individual with an autoimmune disease characterized by dysfunctional expresslon of a transplantation antigen is provided, comprising administering to that individual an oligonucleotide capable of binding to a portion of the transplantation 20 antigen nucleotide sequence, in an amount sufficient to ;
inhibit expression of the transplantation antigen.

Brief Description of the ~rawinas Figure 1 shows the DNA sequence for the X and _ X, boxes of the DR A promoter, and the structure and binding pattern of the triplex-forming oligonucleotides T~ and T2.
~ Figure 2 shows the fluorescence profile of HeLa cells incubated with gamma interferon and various amounts of T~ and then labelled with anti-DR monoclonal antibody.
- Figure 3 shows the fluorescence profile of HeLa ce-lls incubated with gamma interferon and various amounts of Tt and then labelled with anti-DP monoclonal antibody.
_ _ _ - = _ ~ Figure 4 is a Northern blot analysis usinq an anti-sense RNA probe that specifically binds to sense DR

W~93/14769 PCT/US93/007~7 ~ 8 A mRNA. Cells were blotted at 3 and 7 days with the indicated treatments (C.O. indicates control oligonucleotide).
Figure 5 contains the nucleotide sequences for the X and X2 promoter regions for various transplantation antigens.
Figure 6(a) shows the fluorescence profile of gamma interferon induced Colo 38 cells incubated with control antibody (mouse IgG2) and anti-DR monoclonal antibody.
Figure 6(b) shows the fluorescence profile of gamma interferon induced Colo 38 ce~ls treated with ta) nothing, (b) 50 ~M oligo A, or ~c) lOo ~M oligo A, and followed by incubation with anti-DR monoclonal antibody.
Figure 6(c) shows the .luorescence profile of gamma interferon induced Colo 38 cells treated with (a) nothing, or (b) S0 ~M control oligo Al and ~ollowed by ~-incubation with anti-DR monoclonal antibody. ~ ~-Figure 7 shows the fluorescence profi}e from flow cytometry of HeLa cells incubated with gamma interferon and various amounts of TSl and then labeled with anti-DR -monoclonal antibody fluorescein.
Figure 8 shows the Dose Response Percent suppression of cell surface DR antigen as a function of concentration of TSl as it affects ~eLa cells.
Figure 9 shows the duration of TSl effect on HeLa cells. `
Figure lOa shows the efféct of TSl on constitutive DR Colo cells. Figure lOb is the 30 ! accumulative integration along the fluorescence axis of ~-~
the data shown in Figure lOa. ~-Figure ll is a bar graph that shows the effect of anti-sense oligonucleotides ANTI-B, AR, ACAT, ATCT and T2 on the induction of MHC Class I antigen expression by IFN~

W093/14769 ~ PCT/US93/00797 _9_ ,:
Figure 12 is a graph showing the effect of T2 and of A3 on the I~N-~ mediated enhancement of tryptophan degradation.
Figure 13 is a graph showing the effect of oligonucleotides T~ and A3 on kynurenine production.
Figure 14 is a bar graph showing the effect of T2 on HLA Class I induction by IFN-~, IFN-~, and IFN~
Figure 15 is a bar graph showing the effect of - T2 on IFN-~ induced MHC-II in WEHI-3 cells.
Figures 16A and 16B are graphs showing the effect of T2 on IFN-~ and TNF-~ induced ICAM-1 cell surface expression, respectively. I -Figure 17 is a graph~showing the effect of T2 ~
on antigen-induced proliferation of human monocytes. ~`
Figure 18 is a graph showing the effect of T
on T cell actlvation using~an IL-2 production assay. ~-~

Detailed Descri~tion of the Invention The~praotic-~of the~pr-sent inventlon encom-passes conventional~techniques of chemistry, molecular -biology, biochemistry, protein chemistry,~and recombinant DNA technology, whic~ are within the s~ of the art.
Such techniques~are explained fully in the literature.
See, e.~ Oli~onu~cleotide Synthesis (M.J. Gait ed. -~1984);~Nucleic~Acid~H~bridization (B.D. Hames & S.J.
Higgins~ eds~ l98~4)~;~5ambrook, FrLtsch &~Naniatis, Mo-lecul&~-~lonina: A Laborator~ Manual, Second Edition 1989);~PCR-Technoloqy~ (~H~.A. Erlich~ d., Stockton Press);
~ -R.K. Scope,~Protein Purification Principles and Practice 30 ~ ~(Sprihger-~erlag); and the series Metho~s in ~nzymoloaY
(S.~ Colowlck and N. Kaplan eds., Academic Press, Inc.).
- - All patents, patent applications and _p~b~ications mentioned herein, whether supra or infra, are hereby incorporated by reference in their entirety.

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Definitions:
As used herein, the term "transplantation antigen~ is used to refer to antigenic molecules that are expres-~ed 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. The human MHC complex is also called the human leukocyte antigen (HLA) complex. 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 inc~lude the following: (1) .
the ABO antigens involved in blood cell recognition; (2) cell adhesion moIecules such as ICAM, which is involved -in leukocyte cell-cell recognitlon; 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.
As used herein, the term "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 th~ 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 WO 93/14769 ; ^~ ,~ ^? PCl'/US93/00797 found on any form of messenger RNA (mRNA) derived from the gene, including the pre-mRNA, spliced mRNA, and polyadenylated mRNA.
As used herein, the term "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 at all times. Pxeferably, at least 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.
Certain 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-transplan~ 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 -wi^ll survive in the transplant recipient for time periods ~commensurate to norm~l or untreated cells, but will require lower doses of immunosuppressive agents to the transFl~t-recipient.
-- As used herein, "oligomers" or "oligo-nucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. "Nucleic acids", as used herein, re~ers to RNA, DNA, or RNA/DNA hybrid sequences of any =len~th in single-stranded or duplex form.
~ As used herein, the term "binding" refers to an interaction or complexation between an oligonucleotide ~ 12-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.
The second type of binding is "triplex binding"
which follows a set of still-developing binding rules. -In general, triplex binding refers to any type of base-15 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 descri~ed in PCT Application No. WO 90/15884 (published 27 December 1990). In one set of triplex binding rules, the third strand is design~d to match each A or T in one of the duplex strands with T, and each C or G with C, and the third strand runs antiparallel to the ;
matched strand. In another set of triplex rules, the third strand is designed to match each A or T in one of 25 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.
As used herein, Hoogsteen-like bonds refers to hydrogen bonding between bases.

T~e Oliqonucleotide: `
According to this invention, oligonucleotides are synthesized that are capable of binding to a 093/14769 ~ PCT/US93/00797 l3-transplantation antigen nucleotide sequence. The bindin~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.
One specific target sequence is the well-characterized DR A promoter region. The DR A promoter region contains a number of suhregions known to be specific binding sites for DNA binding proteins, called the J, W, X (including X~ and X2), and Y boxes, and the gamma interferon response element. Particularly significant are the X and X2 boxes, as described herein.
Other specific target sequences are within the structure .
gene.
~- In general, a minimum of approximately 5 nucleotides, preferably at least l0 nucleotides, are necess?ry ~o~effect the necessary binding to a specific taFget sequence within the intron region of the structural gene. By targeting the structural gene region, only two target DNA sequences per cell are required to be bound by this oligonucleotide.
Furthermore, short strands of oligonucleotides (a-p~roximately 26 nucleotides or less) are readily taken up by cells. The only apparent limitations on the required binding length of the target/oligonucleotide l ~ ~ ^. r, ~ --14 r~, ., complexes of the invention concern making the oligonucleotide of sufficient binding length to be -~
capzble of binding to the target transplantation antigen sequence, and not to bind to other undesirable non-target sequences and disrupt other cellular mechanisms.
Oli~onucleotides of sequences shorter than 15 nucleotides -may be feasible if the appropriate interaction can be obtained.
As further explained below, the oligonucleotides 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 may also contain "interior -flanking sequences", which are sequences within a binding ``
20 sequence that are not capable of binding to the target -through Watson-Crick or triplex binding rules. Thus, the ~ oligonucleotide may comprise two or more~binding regions separated by nonbinding interior flanking sequences. It ~;
is also contemplated that the bindin~ se~uences may contain one or more mismatches that do not conform to the binding ruIes. These substitutions are contemplated as - ~ part of the invention as long as the oligonucleotide retains its binding capability as described herein.
The oligonucleotide 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) ~ `
239:4~7-491, and European patent applications 86302298.4, 86302299.2 and 87300203.4, as well as Methods in ,;~
EnzymoloqY (1987) 155:335-350. The amplified DNA may , WO93/14769 ~ ' PCT/US93/00797 then be recovered as DNA or RNA, in the original single-stranded or duplex form, using conventional techniques.
The oligonucleotides of the invention usually comprise the naturally-occurrin~ bases, sugars and phosphodiester linkages. However, 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 conjusated to solid supports~ The 5' and 3' terminal OH
groups are conventionally free but may be phosphorylated or substitu~ed with amines or organic capping group-moieties of from 1 to 20 carbon atoms. Other hydroxyls m~y also be derivatized to standard protecting groups.
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 is replaced by P(O)S
("thioate"), PtS)S ("dithioate"), P(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), wherein each R
or R' is independently H or substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in an oligomer need to be identical.
Also included within this invention are synthetic procedures in which the resultant 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 a2iridinylcytosine, 4-acetylcy-tosi-ne,~ 5-fluorouracil~ 5-bromouracil,

5-carboxymethy~laminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, WO93/14769 ~ ~ PCT/US93/00797 ~ ,,..~
l-methyladenine, l-methylpseudouracil, l-methylguanine, l-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N~-methyladenine, 7-methylguanine, 5-methylaminomethyl- ~
5 uracil, 5-methoxyaminomethyl-2-thiouracil, ~ -beta-D-mannosylqueosine, 5-methoxyuracil, 2-methyl-thio-N6-isopentenyladenine, uracil-5-oxyacetic acid ~
methylester, pseudouracil, queosine, 2-thiocytosine, -5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, 5-pentynyluracil and 2,6-diaminopurine. The use of 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 may contain analogous forms of ribose or deoxyribose sugars that are generally `~
known in the art. An exemplary, but not exhaustive Iist -includes 2' substituted sugars such as 2'-O-methyl-, 2'~
O-allyl, 2'-f}uoro- or 2'-azido-ribose, carbocyclic sugar 20 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.
Although the conventional sugars-and bases will be used in applying the method of the invention, substi-; tution of analogous forms of sugars, purines and pyrimidines can be advantageous in designing the final ~;
- product, as can alternative backbone structures like a polyamide backbone.
Oligonucleotides containing the designed binding sequences discerned through the method of the - invention can also be derivatized in various ways.
~~- Primarily, the oligonucleotides will be derivatized by attaching a nuclear localization signal to it to improve targeted delivery to the nucleus. One well-characterized , ~,, ~ ~ P~T/US93/00797 WO93tl4769 -17-nuclear localization si~nal is the heptapeptide PKKKRKVtpro-lys-lys-lys-arg-lys-val). Also, if the ~ oligonucleotide is to be used for separation of the target substance, conventionally the oligonucleotide will be derivatized to a solid support to permit chromato-graphic separation. If the oligonucleotide 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 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.
One desired function that may be performed by the oligonucleotide at the targeted site is alteration of the targeted DNA. The oligonucleotide may be derivatized :
to attach to the targeted sequence, to crosslink t~e targeted sequence (e.g., through psora~en crosslinks), or -to alter, modify or delete all or par~ of the targeted sequence. In-~his-manner, the oligonucleotide may cause a permanent depletion of a transplantation antigen on a -- cell and its daughter cells.
The oI~igon~cleotide sequence may also be included in a suitable expression system that would provide in situ generation of the desired oligonucleotide. ---.
The Methods~
_ _ .. .
According to this invention, theoligonucleotides described above are used in a method of WO 93/ 1 4769 r 1 ~ 1 8 PCr~ US93/00797 treatment to make a transplantation antlgen-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.
Alternatively, the oligonucleotides can be -modified or co-administered for targeted delivery to the ~
nucleus. The cell nucleus is the 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. Also, 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 ~olume; t3) higher concentrations of key enzymes such as RNase H implicated in the mechanism of action of these oligonucleotides. The cytoplasm, however, 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 derlvatizing the oliqonucleotides by attaching a - nuclear localization signal. One well-characterized :
~ 25 -nuclear localization signal is the heptapeptide ~kKKK~
(pro-lys-lys-lys-arg-lys-val).
-- - Any transplantable cell type is a potential ~arget cel} for this invention. Preferably, the target cell is selected from corneal endothelial cells, thyroid icells, 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 -~--35-- muscle cells, and hair follicle cells. Most preferably, the target cell is selected from corneal endothelial ~', J , ~
W O 93/14769 ~ P ~ /US93/00797 cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, ~one marrow cells, pancreatic islet cells and hepatic cells.
In another aspect of this invention, the above-described oligonucleotides may be incorporated into anexpression 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. In this fashion, the desired o}igonucleotides 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 followin~ transplant.
Furthermore, this invention is applicable to the field of solid organ transplants. Organs are normally perfused ex vivo prior to transplantation. By adding an amount of the above-described oligonucleotides to~the perfusion medium, 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.
Finally, local administration of the anti-gene ~ oligonucleotides directly into the transplanted organ ;~ during the first day after the transplant is within the scope of this invention.~Alsor sustained releases of the of these drugs are also contemplated.
, Methods of Treatment and Administration 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 wi~ an immune system, including humans.

WO93/14769 ; ~ PCT/US93/00797 '~:'~..
The oligonucleotides of this invention are also .
useful in treating autoimmune diseases characterized by dysfunctional or aberrant expression of a transplantation ~
antigen. In such a case, the oligonucleotides described -5 herein may be administered in an amount sufficient to ~-`
inhi~it expression of the transplantation antigen. -It may be commented that the mechanism by which the oligonucleotides of the invention interfere with or inhib~it the production of one or more transplantation antig~ens~ is not always established, and is not a part of the invention. The oligonucleotides of;the invention are characterized by their capa~ility to bind to a specific :
target nucieotlde~s~equence regardless of the~mechanisms of binding or the mechanism of the effect thereof.
lS Described below are exampIes of the present ~
inven~ion which are~provided~ for illustrative purposes, and~not to;l~imit the scope ofj~;the present invention. In light of~the~disclosure, numerous~emb~diments within the scope of~the~claims~will be~apparent to those of ordinary 20 ~ ~skiIl in the art.

ples ~ -Materia~ls and Methods 2~5~ ~Cell S~trains~and~Culture Medi~
HeLa S3~cells~(human cervical carcinoma cell ine-aTCC~CCL~2.2),~K~562 cell~`lines~(UCSP Cell Culture Fac~lity),~a~nd~JAB~cells ~(human lymphablastoid cell line, UCSF Cell Culture FaciIity) and Colo 38 (human cervi-cal carcinoma cell }ine) are grown in RPMI 1640 ~ , . .
medium (Gibco) supplemented with 10% fetal calf serum heat inactivated at 65C for 30 minutes. Fibroblast 143B
c~ s--(human osteosarcoma cell line ATCC/crl 8303) is ~; grown in MEM Eagle's BSS medium (UCSF Cel} Culture ~:~ :' '.

r " ~

Facility) supplemented with 10% fetal calf serum heat inactivated at 65C for=30 minutes.

Oliqonucleotide desiqn Phosphodiester oligonucleotide A consisting of 1~ nucleotides was designed to base-pair in Watson-Cric~
fashion to the mRNA base se~uence of the DR A structural gene beginning 13 nucleotides upstream (5') of the translational initiation codon (AUG~, lo A: 5' GCC ATT TTC TTC TTG GGC G 3' and ordered from the UCSF Biomolecular Resource Facility.
Two control phosphodiester oligonucleotides (Al and A2), consisting of random sequences of the same base composition as above but that would not bind to DR A mRNA
were also ordered from the Rescurce Facility:
Al: 5' TTG CCA GAC TAT TGT CCC A 3' A2: 5' TAT CGG CTT TGT TGC CCG T 3' ~-Triplex-forming oligonucleotides (TFOs) were designed to match the HLA DR A X and X2 box promoter. T~, T2, T~C and T~ were ordered from American Synthesis Inc..
T~, T2, T2C were designed according to the formula shown in Figure 1. T~ was designed to have a C to match each GC base pair in the duplex and a T to match each AT pair.
T, was designed to have a G to match each GC base pair in the duplex and a T to match each AT pair. Tl and T2 were modified with a 3' amino group to increase stability~
T2C is the same as T2 except T2C-is- unmodified. T, was designed as a control oligonucleotide with the same overall nucleotide composition as T2 and T2C but with its sequence altered to havè less triplex-type pairing with the X and X~ boxes:
T~: S' TGT TGG TGT GGG TTG TGG TTG GTT GC 3' _ Al and A2 are unmodified~ollgonucleotide se~uences that would not form triplex structures with the promoters.

W O 93/14769 P ~ /US93/00797 . .:
TS1 is a 26 nucleotide oligonucleotide consisting ~ of a phosphodiester backbone and amine modified 3' ~-terminus. T~1 was designed to be anti-parallel to the coding strand with the maximum number of Hoogstein bonds ;~
that can form between TS1 and the targeted sequence.
Under physiologic conditions the formation of GGC and TAT
bare triplets are favored, giving rise to a triplex helix with one DNA strand of the DR A gene at residue positions 5'-851 to 3'-876 as numbered in Sch~rhoeck, A., et al., Nuc. Acids Res. (1983) 11:8663-8675. TS1 is unique in that it will bind either parallel or antiparallel because of the palindrome character of duplex DNA. The DR A gene was selected because it is monomorphic between individuals, thereby minimizing the variability of gene sequence which normally occurs in polymorphic genes. The method to construct the TS1 sequence was to select a C
for every C or G in the DNA target sequence and a T for every A or T. Tc~ and GTC~ are control oligonucleotides ~-that are also 26 nucleotides in length and amine modified ~
20 at the 3' terminus. The sequences are compared to the ~ i segment of the DR A intron of the structural gene below:
DR A: 5'-GGG GGT GGG GGT GGG GGT GGG GGA GG-3' ~
TS1: 3'-GGG GGT GGG GGT GGG GGT GGG GGT GG-5' -Tc~ 3'-TTT GTG TTT TGT TTT TTT GTT TTT TT-5' -25 - GTC~: 3'-GGT GTG TGT GTG TGT GTG TGT GTG TG-5' '~
The oligonucleotides were ordered from Keystone - ~-L~boratories.
~ Oligonucleotide A3 is a control oligonurleotide that showed relatively lower ability to inhibit the IFN-~ ~
30 enhanced M~C-I expression in HeLa S3 cells. The X at the ~-end of the oligonucleotide represents the 3'-amino linker ... .
modification discussed for T2 oligonucleotide. The -- seguence of A3 is the following.
A3: S ' TTG CCA GAC TAT TGT CCC A X 3 ' : -;

W093/14769 ~`~ PCT/US93/00797 Other oligonucleotides used are the following.
Oligonucleo~ide CL~--is designed to be antisense to the ATG site in HLA-A2 mRNA. Oligonucleotide ANTI-B is designed to be identical to one strand in the KB~ binding site in the enhancer A region of the MHC-I HLA-A2 promoter. Oligonucleotide AB is directed toward the Enhancer B in the 5'-region of MHC-I A2 gene. ACAT is an 18 mer directed towards the CAAT ~ox of the MHC-I A2 gene. ATCT is directed towards ths MHC-I A2 e~uivalent of the TATA box. These oligonucleotides and the A3 oligonucleotide were purchased from Key~tone Laboratories, Menlo Park, CA. The sequen es of these nucleotides are the following.
CL~: 5' AGG GTT CGG GGC GCC ATG ACG GC X 3' ANTI-B: 5' CCC AGC CTT GGG GAT TCC CCA AC~ CC X 3' AB: 5' CCG ACA CCC AAT GGG AGT X 3' ACAT: 5' CGA CAC TGA TTG GCT TCT 3' ATCT: 5' TGC GTG CGG ACT TTA GAA X 3' Antibodies Mouse Anti-human HLA-A,B,C, anti-~2 microglobulin and control IgG2b antibodies were purchased as fluorescein isothiocyanate conjugates from Olympus, Lake Success, N.Y.. Mouse anti ICAM-1 and IgG~
antibodies were purchased from AMAC, Westbrook ME as fluorescein isothiocyanate conjugates.
- =
Oliqonucleotide U~take and-Gamma Interferon Addition Oligonucleotides were added to the cell medium as described by Orson et al., Nuc. Acids Res. (1991) 19:3435-3441. Cells were initially concentrated to 2-6 x 10~ cells/ml and incubated with various oligonucleotides at various concentrations-~5~M, lO~M, 20~M) with gentle shaking every 30 minutes for 2 hours. Cells were then wo93/1~76s PCTtUS93/00797 ~ r J 24 diluted to 0.2 x 10/ml in order to optimize cell growth.
- At Days 1 and/or 2 cells were again concentrated to 2-6 x 106 cells/ml, and incubated with the oligonucleotide and diluted as above. Where appropriate, gamma inter~eron (IFN~, Collaborative Research, Inc.) at 200 units/ml was added at Days 0, 2, 4 and 6 to induce HLA-DR expression.

Cell surface HLA detection. -At days 3 and 7, cells were isolated and stained with fluorescein isothiocyanate conjugated tFITC) anti-HLA-DR monoclonal antibodies. HLA-DP was detected by indirect staining with mouse anti-DP monoc~onal antibody followed by FITC goat anti-mouse (Becton- -Dickinson). About 0.1 x 10~ cells were used per assay. -10 ~l of mouse IgG2-~ITC served as background control and 10 ~1 of monoclonal anti-DR-FITC IgG2~was added to detect cell~surface HLA DR expression. Monoclonal antibody was incubated with cells on ice for 30 min. The mixture was --;
then washed~with phosphate ~uffered saline (PBS) with 0.1% sodium azide, the supernatant was removed after centrifugation, and the pellet was resuspended in lS0 ml ~;
of PB~S and 50 ul of 0.05% propidium iodide. Flow cytometry analysis (FACScan - Becton-Dickinson) was used to d~tect~cell surface ant-igen expression. A similar 2S procedure usin~ anti-HLA-DP specific monoclonal antibody was used to measure surface HLA-DP expression.
. ..
-Northern Hybridization and RNA Isolation Approximately 2 x 106 cells treated with 30 oligonucleotide were isolated at Day 3 and Day 7. The `~
- - cel-ls~were washed with cold PBS and resuspended in 200 ml ..
cold lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 10 mM
_ ~- Trls-pH 8.0, 2 mM MgCl). Cell nuclei were removed by spinning at 15,000 RPM for S min. An equal volume of protein denaturing buffer (10 mM EDTA, 450 mM NaCl, 7 M

. . . .
' WO93/14769 ~ PCT~US93/00797 urea, 10 mM Tris pH 7.4, 1% SDS) was added to the supernatant. The resulting solution was extracted with equal volumes of phenol/chloroform and the aqueous phase transferred to 0.1 Yolumes of 3 M sodium acetate (pH 5.2 and 2 volumes of 100% ethanol. The solution was kept at -200C overnight. Supern~tant was removed after centrifugation and the RNA pellet was washed with 1 ml 70% ethanol. ~NA wa~ then dissolved in 50~1 of TE buffer (10 mM Tris pH 7.4, 1 ~M EDTA). RNA concentration was ascertained by optical density at 260 nm ~OD2~).
Plasmid containing the DR A gene (DR A PBS M13) was obtained from Lars Karlsson at the Scripps Research Institute, and linearized by incubation with EcoRI. T3 RNA polymerase was added along with ATP, CTP, GTP, and digoxigen-coupled UTP to synthesize DR A RNA probes. The resulting antisense DR A RNA probe was used to detect sense DR A mRNA. A similar method was used to prepare the control antisense ~ actin RNA probe.
7 to 10 ~g of the prepared RNA extract were separated by 1.2~ agarose formaldehyde gel along with ~NA
size markers (Pharmacia or Gibco BRL) at 125 volts for 5-

6 hours. Separated RNA in the gel was blotted onto nylon paper overnight, and baked at 80C for 2 hrs. The paper was then put into a prehybridizing solution (Genius protocol) for 6-8 hours and hybridized with antisense DR
A RNA probe (l~g/ml) and antisense ~-actin probe (0.5 ~g/ml) for about 48 hours.- Anti--d~goxigen alkaline phosphatase-conjugated monoclonal antibody was added to the blotted paper, followed by Lumi-Phos 530 (Boehringer Mannheim, Indianapolis, IN). Lumi-Phos light emission was de~ected by autoradiography.

RNase Protection AssaY ~~
32P-Riboprobes were generated using a MaxiScript in vitro transcription kit (Amblon, Austin TX) and alpha WO 93/14769 PCI'/US93/00797 ; . ' i~ ~ , ;~ --2 6--.. ;.~ :......

3~P-UTP (NEN Dupont, Boston MA). Analyses of RNA were pe~formed using an RNAse protection assay kit tAmbion, Austin, TX). A 0.16-1.77 kb RNA ladder (Gibco, Grand Island, NY) was dephosphorylated using calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN), 32P-labeled with gamma 32P-ATP (NEN Dupont, Boston, MA) and used as molecular weight markers in RNAse protection assays. Isolated RNA from HeLa S3 was hybridized to a T3 polymerase generated 32P-RNA probe antisense to a 1.2 kb fragment of HLA DR A gene (same fragment as used in Northern analysis) probe protected - from RNAse digestion after hybridization to HLA-DR A mRNA
was recovered and electrophoresed on a denaturing 7% ~`
polyacrylamide gel. Each RNA sample from HeLa S3 was simultaneously hybridized to a T3 polymerase generated 32P-riboprobe antisense to a glyceraldehyde-3-phosphate --dehydrogenase (GPD) transcript (0.14kb). Hybridization with the GP~ probe was used to compare amounts of initial total ~NA in the samples analyzed by RNAse protection.
Detection of ICAM-l The presence of ICAM-1 sites on cells was determined as follows. Samples containing O.lml of cells <~
and reactants were drawn from each tube at various times and stained for flow cytometry using anti-ICAM-l antibody. The cell suspension was washed once with 0.75 ml p~osph~te buffered saline (PBS) containing 2% fetal :
calf-serum and 0.1% sodium azide. The antibody conjugate (lO~g) was added and the mixture was agitated. The cells were incubated for 30 minutes on ice in the dark, washed , twice~`-with 0.75 ml of PBS containing 0.1% sodium azide to remove unbound antibody, and resuspended in O.lml PBS
con~a~~n~ng~ sodium azide (0.1%). Propidium iodide was 3S added to exclude dead cells from the analysis. The mean number of ICAM-1 sites was estimated by first determining W O 93/14769 ~ i 3 ~ ~ P ~ /US~3/00797 -~7-the fluorescence to antibody (F/P) ratio for the ICAM-l antibody and its cognate IgG~ on~-~Simply Cellular beads (Flow Cytometry Standards Corporation, Research Triangle, N.C.) containing a fixed number of goat anti-mouse sites.
The flow cytometer was calibrated with QuickCal beads (Flow Cytometry Standards Corporation, Research Triangle, NC). The calibration curve was used in conjunction with the F/P ratio to estimate the mean number of ICAM-l sites from the mean channel fluorescence according to the manufacturer's directions.

Indoleamine 2 3 dioxy~enase assay Indoleamine 2,3 dioxygenase (ID0) of cells was estimated using a spectrophotometric assay. ID0 converts tryptophan to kineurenine. Tryptophan was measured at its absorption maximum of 280 nm, a wave length at which kynurenine does not absorb significantly. Kynurenine was measured at 360;nm, a wave length at which tryptophan does not absorb significantly.
ExampIe l -~
~ wo 26~base pair ~bp~ triplex-forming oligo-nucleotides (TFOs) were designed. Oligonucleotide T~ was - designed by using a T to match each A-T bp and a C for each G-C bp. Oligonucleotide T2 was designed by using a T to match each A-T bp and a G for each G-C bp. Each oligonucleotide was modified with a 3~ amlno group to prolong its half-life. ~In each experiment He1a cells or fibro~last 143B cells were pre-incubated with T~, T2, or T~ for 2 hours before exposure to 200 lnits of recombinant gamma interferon, which was added on day 0 and day 2 of culture. Tl and T2 were also-added on day l _ and day 2. Induction of DR express-ian--was then measured on day 3 by flow cytometry using anti-DR monoclonal antibody which binds to those HLA class II antigens on ~ ~.;. ,` 28-~ . ... ` ~
the cell surface. Although cells cultured with the control ~FO T~ showed normal induction of HLA-DR and DP
antigens, T~ inhibited expression of D~ and DP by 50%, ;~
while T~ showed about 100% inhibition of both DR and DP
at 20 ~M concentration (Figs. 2-3, T~ data only). At lower doses of oligonucleotide (5 and 10 ~M~, both T, and T~ showed inhibitory effects in a dose responsive fashion.
The mechanism of this inhibitory effect was lo investigated further. Transcription of the DR A gene was measured by Northern blot analysis (Figure 4) using an anti-sense RNA probe that specifically binds to sense mRNA. At a concentration of 20 ~M, T2 completely -suppressed DR A mRNA expression measured at day 3. This 15 suppression was reversible with the continued addition of `~-gamma interferon to the culture, as shown at day 7.
While T~ and T2 were able to block gamma interferon-induced HLA DR and DP expression,;they had no effect on constitutive DR expression. This was proven by 20 treating BJAB (B lymphoblastoid) cells and Colo 38 ` -~
(malignant melanoma) cells, both of which express DR
- constitutively, with 5-20 ~M of T~ and/or T2 for up to 7 continuous days. No decrease in the constitutive expression of celI surface DR was observed.
The-effects of T2 appear to be somewhat specific for promoter sequences that share homology with HLA-DR A (Figure--5);~~ ICAM-l, an adhesion molecule which is constitutively expressed on HeLa cells, can be increased by gamma interferon treatment. T2 blocks the augmentation of ICAM expression but leaves the constitutive expression intact and has no effect on either constitutive or inducible expression of HLA Class i ~-- _ I genes.

.

Example 2 The above protocols were fo~lowed to test the antisense nucleotide A and controls A1 and A2 with the following differences. Colo 38 cells were subcultured at 0.1 to 0.3 x 106 cells/ml and incubated with various oligonucleotides at concentrations of 1 - 100 ~M (added `
twice a day, approximately 9 a.m. and S p.m.), presuming complete depletion prior to addition. Gamma interferon `
(Collaborative Research, Inc.) was added at 2Q0 units/ml ~
10 on days 0 and 2 to induce DR A expression and the cells ;-were harvested for flow cytometry analysis on day 3. -Results are shown in F~igure 6. Figure 6(a) shows that~qamma interferon induced cells~are specifically bound by DR specific antibody. Figure 6(b) indicates that increased levels of added oligo A reduces the~amount of specifically bound antibody, si~nifying decr~D~expression of DR A antigen. Figure 6(c) shows that~the addition of control oligos A1 or A2 does not reduce~DR A antigen expression.
amDle 3 The above protocols used for oligonucleotides ;T~and Tz;~wére follQwe~d to test the~antisense oligonucleotide TSl~and controls~Tc~ and GTc~, except that 25 ~ the~fo~ owing differences were;used~. The-oligonucleotides were~ added to the media for 3 days in the cas~e of HeLa cells, and for 5 days in-thè case of other type~s of cells. Gamma interferon was~added to the HeLa and kera~lnocytes cells on days 0, 2, 4, and 6. No gamma interferon was added to the Colo cells~ TSl was added daily to all the cells at 20~M-except for the dose ~-~ response experiment; in the dose rèsponse expe-riment, the TSl concentration varied from 0.1 to 4~-~M.--- ~ The TS1 dose responsiveness of HeLa cells as indicated by the binding of fluorescent anti-DR A

~ ,.
.'`

::`

` -i 2 ~" .~.
monoclonal antibody is shown in Figure 7. Figure 8 compares the dose~response of HeLa cells to TS1 with that to the control oligonucleotide GTC~. As indicated in ~oth Figure 7 and Figure 8, TS1 at levels of 5 ~M and 10 ~M give greater than fifty and ninety percent inhibition, respectively, of the expression of the DR A antigen. '~
The duration of TS1 inhibition of DR A ~
expression in HeLa cells is shown in Figure 9. The HeLa ~-cells were treated with ~00 units/ml gamma interferon on days 0, 2, 4, and 6. 20 ~M TS1 or GTC~ was added on days 0, 1, and 2. On days 3 and 7, the cells were treated with anti-DR monoclonal antibody-fluorescein and analyzed by flow cytometry. Figure 9 shows that TSl completely ' ' suppresses the surface expression of DR after 3 days and lS 7 days. The effect of TS1 remains at least 4 days after it lS removed from the media. Unlike T2 where the cells ,~
begin,to resynthesize DR after removal of the oligonucleotide, TS1, has a longer lasting effect.
The effect of TSl on the constitutive --20 expression of DR expression on Colo 38 cells was also ;~
examined. The results on the binding of anti-DR
monoclonaI antibody-fluorescein to TS1 treated and Tcon ~' treated cells is shown in Figure 10. Figure 10 depicts ~-~
the flow cytometry data when cells are treated with Tcon (Peak B) and TS1 (Peak-A). Peak B has been mathematically reduced so that its peak height coincides with the right ~, - peak height of curve A-.- Pea~ C is the mathematical result ~'' of subtrac~ing reduced Peak B'from Peak A. Figure lOb (Percent vs. Fluorescence) is the accumulative ~` 30 integration along the fluorescence axis of Peak C. This figure indicates that`TSl decreased the expression of cell surface DR antigen in part of the cells, as represented by,the-sha~ed:area. This shaded area represents approximately 20% of the cells, based upon the , integration curves in the lower figure. TSl has a partial ~'~

effect in constitutively expressed DR antigen, but the treatments have not been ~ptimized yet.
To further elucidate the mechanism of action of TSl, DR A RNA levels were determined in untreated, TS1 treated, and Tcon treated Colo cells. RNA was extracted and incu~ated with a 32P-DR A probe~ The probe was prepared from a plasmid containing the DR A gene (DR A
PBS M13~ obtained from Lars Karlsson at the Scripps Research Institute, and linearized by incubation with EcoRI. The RNase Protection Assay was previously described on page 23.
Table 1 TS1 EFFECTS ON CONSTI~ V~: DR A RNA
RNase Protection Assay CPM %Control Untreated DR A 423 ~5%

20~M TSl DR A lS8 22%

20~M Tc~
DR A 158 52%

The G3PDH (glycerol aldehyde-3-phosphate-dehydrogenase) probe was used as a control to determine the levels of RNA loaded onto each gel lane. When-the-RNA
is loaded unequally into each lane, the labelled-bands-for each probe can be excised and counted. Normalizing to the G3PDH radioactivity allows rough comparison of the DR
A RNA. Table 1 shows that when DR A results are~
normalized to G3PDH, 20 ~M TS1 decreased the DR A RNA
level by approximately 50~ when compared tQ un~reated or Tcon treated cells.

. .

WO93t14769 PCT/US93/00797 The cross reactivity of TS1 to other gamma interferon genes (I~-N-~) was determined in HeLa cells.

Table 2 ~;~
TS1 SPECIFICITY - HELA CELLS ~;
Mean Channel Shift tEx~erimental - Control) DR DP ICAM CLASS I
Untreated 0 - 0 54 111 IFN-~ 21 13 119 141 20~M TS1 o 0 63 125 20~M Control 19 7 119 13 ~GTC~ or ~;
Tc~) . ' Table 2 shows the cross reactivity of TS1 to other gamma interferon genes. TSl completely suppresses `~
gamma interferon induced DR and DP expression. TS1 ;~
suppresses gamma interferon ICAM suppression~to levels of P
constitutive expression. It is unknown whether TS1 reduces constitutive as well as gamma interferon induced - ICAM antigen. TSl has very little effect on HLA Class I
expression, both constitutive and gamma interferon induced.
~he effect of TS1 on keratinocytes, a type of "normal" primary cell, not an immortal cell line, was also evaluated. --~~~

:.

WO93/14769` PCT/US93~00797 Table 3 TSI EFFECT ON KERATINOCYTES
Mean Channel Shift (Ex~erimenta} - Control~
Untreated 6 IFN-~ 31 20~M TSl 0 20~M Tc~ ll Table 3 shows that TSl completely suppresses the gamma interferon inducible DR antigen expression on ~eratinocytes. T~ also shows some suppression.
In summary, these results show that, in contrast to the earlier studies with oligonucleotide T2, treatment with TSl inhibits the transcription and expression of constitutively synthesized HLA DR A
antigens. In addition, TSl effects appear to have longer duration than those of Tl.
!
Exam~le 4 The above prQtocols were followed to test the effect of anti-sense nucleotides on the induction of MHC
Class I antigens by IFN-~ K562 cells were treated with ; 25 ~M of the following o}igonucleotides for 2 hours prior to the addition of 500 U/ml of IFN-~: ANTI-~, A8, ACAT, ATCT and T2. Fresh Qligonu~leotides were added~at 24, 48, 72, and 92 hours after the addition of IFN-~, and the cells were analyzed by antibody staining and-flow - cytome~ry at 100 hours for the presence of MHC~
antigens. The results presented in Figure ll show that 30 the anti-sense nucleotides tested are all capable of ~`
preventing the upregulation of MHC Class I antigens by IFN-y.

Exam~le 5 --:, . .
" '.
.;:

WO93/14~69 ~ PCT/US93/007g7 i3 ~ -34-Antiproliferat1ve and antitumor effects of IFN-~ are thought=to be mediated primarily by induction of an enzyme in the tryptophan catabolism pathway, indoleamine 2,3 dioxygenase (ID0). (Taylor and Feng, FASEB J., 5:2516 (1991)). IFN-~ and ID0 induction have been observed in tumor allografts undergoing rejection suggesting that activation of the catabolism pathway may be one of the factors involved in graft rejection.
(Takikawa et al, J. Immunol. 145:1246 (1990)).
The protocols described above were used to examine the effect of oligonucleotide T2 on the induction of ID0 by IFN-~. At 0, 24, and 48 hours HeLa S3 cells in RPMI i640 were incubated for 2 hours at 37C with 25 ~M
of either oligonucleotide Tt or A3 (control) and then stimulated with 500 U/ml of IFN-~. Two aliquots, each containing 5 x 104 celIs were removed; one ali~uot was transferred to Hank's balanced salt solution (HBSS) and the other to HBSS containing 50 ~M L-tryptophan (Sigma, St. Louis, M0). A280 and A3~ measurements were taken at intervals on supernatants from both samples.
Figure 12 is a graph showing the effect of T2 - and of A3 on the IFN-y mediated enhancement of tryptophan degradation. As seen from the results, T2 but not A3 - ~ inhibited the increase in the rate of tryptophan ~25 degradation induced by IFN~
ID0 converts tryptophan to kynurenine. The results in Figure 13 confi-rm-that the decrease in tryptophan is accompànied~by a corresponding increase in - material that absorbs at 360 nm, the wave length at which ~ynurenine absorbs. Figure 13 is a graph showing the effect of oligonucleo-tides-T2 and A3 on kynurenine production. As seen from the graph, T2 but not A3 inhibited the inc~ea-se--in-the rate of kynurenine production induced by IFN-~.

WO 93/14769 r~ . PCr/US93/007g7 ~ 3 5 -Example 6 The above-described protocols werP used to examine the effect of T~ on HLA Class I induction by IFN-~, IFN-~, and IFN-~. The cells were incubated with the interferons indicated in Figure 14, and with 25 ~M T2 or without oligonucleotide ~Control). The amount of MHC
Cla~s I antigens was determined by cell sorting after staining with two antibodies, one directed to the heavy chain and the other directed to ~2-microglobulin~ As seen from the results, shown in the bar graph in Figure 14, T2 inhibits the induction by IFN-~. However, it does not inhibit induction by IFN-~ or IFN-~. In Figure 14 MCS is the mean channel shift and the change from cells not treated with interferon is plotted on the y-axis.

Example 7 The following illustrates that T2 prevents the induction of mouse MHC class~II by gamma interferon.
It has been shown that the promoter region of the mouse MHC displays a larg~ degree of homology with its human counterpart. In order to investigate the effect of T2 on the expression of mous MHC class II
molecul~, we utilized the myelomonocytic cell line, WEHI-_ 3. This cell line derived from BALB/c mice (H-2d) expresses MHC class II molecules (Ad,E~r`at very low levels (4%). However, both Ad and Ed molecules can be induced on these cells following 72 hours trea-tment with gamma interferon. Consequently, gamma Interferon-mediated MHC class II expression on WE~I-3 restores the capacity of these cells to stimulate class II-restricted T cell pro}iferation in an antigen specific manner.
WE~I-3 were preincubated with the oligonucl~otide T2 (final concentration 25, 50, 100 mM~ for ~ hours, then cultured in the presence of gamma interferon (100 U/ml) for 48 hours. Cell surface expression of MHC class II Ad -~

WO93/14769 ~ .'J~J ~' 2 -36- PCT/US93/00797 and Ed was then measured by cytofluorometry analysis (FACS) using di--rect staining with FITC-labelled anti-MHC
class II monoclonal antibodies (I-Ed). Figure lS is a ;
bar graph showing the effect of T2 on IFN-y induced MHC-II in WEHI-3 cells. The results are indicated as mean channel shift corresponding to: number of channel for -anti-I-Ed mAB - number of channels for a control IgG2 mAb.. As seen from the results in Figure 15~, T2, complete}y abollshed the induction by gamma interferon of ~-~
murine MHC class II on WEHI-3~ Gell lines (95% reduction).

Exam~le 8 I ~-The oligonucleotide (T2) designed to form a triplex helix with the promoter region of the human major histocompatibility complex (HLA) locus has been shown to prevent the induction by gamma Interferon of HLA class II
(DR)~ cell surface molecules on different cells (Hela, ; fibrobla~sts, keratinocytes) (See above). The effect of ;~
T2 ~on~thé~surface expression of another gamma interferon-2~0~ inducible~;immune receptor was examined.
Hela cells were preincubated for 2 hours with the oligonucleotide T2 (20uM) or with a~control ` oligonucleotide~(Al)~ or with medium alone. Then, the cells were cultured~for different periods of time in the ';~
presence of~gamma~interferon (50 U/ml)(Figure 16A) or Tumor Necrosis factor~(TNF) a~lpha (150 U/ml)(Figure 16B).
Follow~lng~t:his~st~p, the--cells~were stained with a F~TC- ;~
labelled ant~ M-l and~analyzed by cytofluorometry (FACS).~ The results are indicated as mean channel shift 'corresponding to: number of channel for anti-I-Ed mAB -number of channelS for control IgG2 mAb.
As~seen from the results in Figure 16A, there ~ was a complete s~press on of the gamma Interferon-'~ ~ induced expressi'on~of the adhesion molecule ICAM-1.
:
~ However, as seen from Figure 16B, T2 had no effect on and .

W O 93/14769 P ~ /US93/00797 TNF-a mediated ICAM-l cell surface expression on these human cells. In other studies, a lack of effect was seen both with IL-1 and IL-4 mediated ICAM-l cell surface expression. Therefore, we conclude that the oligonucleotide T2 is specific for gamma Interferon-mediated functions.

Example 9 .
This example illustrates that T7 inhibits T
cell prolifera~ion and IL-2 production by preventing antigen presentation by accessory~cells.
!
a) T2 blocks anti-CD3 medi~ted human T cell ~roliferation bY preventinq t~e exDre~sion of Fc rece~tor~ on monoc~te~
Whether~other gammà interferon-induced immune receptors~would be suppressed by;~T2 and whether this 20 would impact~human T~cell responses was examined. More -speciflcal~ly, ~ the influence of T2 on gamma~interferon-induced expression~of Fc receptors~ on human monocytes, i;
and~2~ the~ca~pacity of T2-treated-monocytes to fulfilI ~-"
25~ ~thelr~accèssory~funcbions such~as supporting nti-CD3-media~ed~human~T cell proliferation were examined. ~;`
It;~is well established tha~t~human T cells ~an-~be stimulated to proliferate when incubated~with-,. ~
monoclonal antibodies (mAb) directed to the CD3 complex OKT3). ~To mediat~e this-effect, anti-CD3 mA~ need-first to bind through their Fc portion~to Fc receptors ~FcR~ on monocytes in order to stimulate IL-l secretion by these .~, : ~ .'.",:

, ` `-s~
~ 38-cells and to aggregate TCR/CD3 complexes on T
lymphocytes. Both signals (IL-l and CD3/TCR aggregation) are necessary to trigger T cell proliferation and IL-2 5 produc~ion. Gamma interferon regulates the expression of -~
FcR on human monocytes. Gamma interferon-mediated induction of FcR on monocytes restores IgG1 anti-CD3 (Leu-4, UCHT-1)-mediated T cell proliferation in non-responder individuals as well as enhances IgG2 anti-CD3 (OKT3)-mediated T cell mitogenesis (G. Benichou et al., Eur. J. Immunol 1987, 17:1175-1181). , ;

In the s~udy, the results of which are shown in ~igure 17, human monocytes were purified by adherence from human peripheral blood mononuclear cells (PBMC).
They were preincubated in the presence of the oligonucleotide T2 at different final concentrations ranging from 5 uM to 50 uM, or in the absence of oligonucleotide (dashed line). Then, the cells were washed and treated f-or-48 hours with different , concentrations of gamma-interferon. Following this step, the mononuclear cells were cocultured with syngeneic peripheral T lymphocytes~in~-the,presence of anti-CD3 monoclonal antibodies (OKT3) in 96-well culture dishes for 4 days. Antigen-indu,ced proliferation was assessed by the incorpora~ion of 1 mCi ~3H]-thymidine during the last 18 hours of cu}t~re.-- Results are expressed as W093/14769 ~ PCT/USg3/00797 counts per minute tcpm) obtained with cells stimulated in vitro with anti-CD3 mAb.
The results of the study indicate that: 1) T2 completely abolished (100% inhibition) the induction by gamma interferon of FcR at the surface of monocy~es; and ~) the lack of FcR rendered human monocytes incapable of supporting OKT3-mediated in vitro T cell proliferation of 1 human T lymphocytes (Figure 17).

b) ~2 inhibitq anti~en-mediated I~-2 relea~e bv mouse T ~ell hYbridomas. ~
The effect of inhibition of gamma interferon- ;:
induced MHC class II on mouse cell~ on antigen presentation for T cell activation was examined. T
lymphocytes recognize the antigen in the form of peptide presented in association with self-MHC molecules at the surface of antlgen presenting cells (APC). Following immunization, CD4+ T helper cells initiate the immune 25 response by interacting through their antigen re~eptor- ~.
(TCR) with the bimolecular complex formed by the MHC
class II and the peptide antigen. Antigen reco~nitlon ~y~ `.
T cells triggers their proliferation and the secretion 30 interleukin-2. Here we tested whether the `
oligonucleotide T2 would influence antigen presentation by a cloned APC, WEHI3. WE~I3 is a myelomonocytic=celI

35 line whose level of MHC expression was very low --~4%3 but -~
could be increased up to 90~ following exposure to gamma ~'093/14769 PCT/US93/00797 ~ 40-interferon. We have shown that the induction of MHC
class II (Ad,Ed) on WEHI3 by gamma interferon can be blocked by preincubating the murine cell line with T2 (20 S mM final concentration). In order to measure the effect on T activation we used a T cell hybridoma, lEl specific for the lambda repressor peptide 12-24 presented in association with the murine MHC class II molecule, Ed.
Following preincubation with gamma interferon, WEHI3 displays high levels of surface Ed molecule and presents efficiently the peptide to the T cell hybridoma, lEl.

15 The oligonucleotide T2 suppressed antigen presenting ~ `
functions of WEHI-3 in that it prevented the in vitro interleukin 2 (IL-2) production of the CD4+, class II-restricted T cell hybridoma (lEl) to its specific antigen, the lambda repressor peptide 12-24 (Figure 18).
In the study for Figure 18, l x lOs lEl T
hybridoma cells specific for the lambda repressor peptide, 12-24, in association with I-Ed were used. They were cocultured ~or 24 h with the A20 (Ad,Ed) B cell lymphoma ( 105 cells) as APC control, or with WEHI-3 myelomonocytic cell line treated~with gamma interferon ~lO0 u/ml) in the presence of the oligonucleotide T2 or with medium alone. Then the relevant peptide was added to the cell culture at different concentrations. The 96-well microplates were then-ce~trifuged, and the culture supernatants (lO0 ml) were aspirated and transferred ~o a WO93/14769 ; ,~ ~1 PCT/US93/00797 -4l-new microtiter tray which was frozen and thawed before assay for IL-2 production. IL-2 was assayed by [3H]-thymidine incorporation of the IL-2-dependent cPll line, HT2. Briefly, 0.04 ml of culture supernatants were further incubated with 104 HT-2 for 24 h in a total :~
volume of 0.2 ml HL-l medium. Incorporation of l mCi [3H]-thymidine was assayed during the last 4 h of ~.
culture.
The results from the studies indicate that the oligonucleotide T2 can block in both human and murine ~.
systems, the induction of different cell surface -:-:
receptors by gamma interferon, but not other lymphokines.
This results in the abolition~ of the capacity of a murine .-cell line to present the antigen and to stimulate in ;~
20 vitro antigen specific T cell proliferation. ~:~
'' 2s - ~
"";

;

-_

Claims (33)

Claims We claim:

1. A method for making a transplantation antigen-depleted cell from a target cell, the method comprising:
(a) obtaining the target cell; and (b) exposing the target cell to an oligo-nucleotide capable of binding to a transplantation antigen nuclestide sequence, said oligonucleotide being present in an amount sufficient to make the target cell a transplantation antigen-depleted cell.

2. The method of claim 1 wherein the oligonucleotide is capable of binding to the transplantation antigen nucleotide sequence through Watson-Crick binding or through triplex binding.

3. The method of claim 2 wherein the transplantation antigen nucleotide sequence is a ICAM
gene sequence or an MHC class I gene sequence or an MHC
class II gene sequence.

4. The method of claim 3 wherein the MHC class II gene sequence is located at the DR A, DP, or DQ locus.

5. The method of claim 4 wherein the MHC
class II gene sequence is located at the DR A locus.

6. The method of claim 5 wherein the oligonucleotide comprises the sequence 5' GCC ATT TTC TTC TTG GGC G 3' or fragments thereof which retain binding capability.

7. The method of claim 2 wherein the transplantation antigen nucleotide sequence is a promoter sequence or a structural gene sequence.

8. The method of claim 7 wherein the transplantation antigen nucleotide sequence is a DR A
structural gene sequence or a DR A promoter sequence.

9. The method of claim 8 wherein the DR A
promoter sequence comprises the X-box or X2-box.

10. The method of claim 9 or claim 1 wherein the oligonucleotide comprises the sequence 5' GGGGTTGGTTGTGTTGGGTGTTGTGT 3' or fragments thereof which retain binding-capability.

11. The method of claim 7 wherein the oligonucleotide comprises the sequence 5' GGTGGGGGTGGGGGTGGGGGTGGGGG 3' or fragments thereof which retain binding capability.

12. The method of claim l wherein the target cell is selected from the group consisting of corneal endothelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells and hepatic cells.

13. The method of claim 1 wherein the transplantation antigen nucleotide sequence is an ICAM
gene sequence.

14. A transplantation antigen-depleted cell prepared by a method comprising:
(a) obtaining a target cell; and (b) exposing the target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, said oligonucleotide being present in an amount sufficient to make the target cell a transplantation antigen-depleted cell.

15. The transplantation antigen-depleted cell of claim 14 wherein the oligonucleotide is capable of binding to the transplantation antigen nucleotide sequence through Watson-Crick binding or through triplex binding.

16. The transplantation antigen-depleted cell of claim 15 wherein the transplantation antigen nucleotide sequence is a ICAM gene sequence or a MHC
class I gene sequence or a MHC class II gene sequence.

17. The transplantation antlgen-depleted cell of claim 16 wherein the MHC class II gene sequence is located at the DR A, DQ or DP locus.

18. The transplantation antigen-depleted cell of claim 17 wherein the MHC class II gene sequence is located at the DR A locus.

19. The transplantation antigen-depleted cell of claim 15 wherein the transplantation antigen nucleotide sequence is a promoter sequence or a structural gene sequence.

20. The transplantation antigen-depleted cell of claim 19 wherein the structural gene sequence is a DR
A structural gene sequence or the promoter sequence-is-a DR A promoter sequence.

21. The transplantation antigen-depleted cell of claim 20 wherein the DR A promoter sequence comprises the X-box or X2-box.

22. The transplantation antigen-depleted cell of claim 21 or of claim 14 wherein the oligonucleotide comprises the sequence 5' GGGGTTGGTTGTGTTGGGTGTTGTGT 3' or the sequence 5' GGTGGGGGTGGGGGTGGGGGTGGGGG 3' or fragments thereof which retain binding capability.

23. The transplantation antigen-depleted cell of claim 14 wherein the target cell is selected from the group consisting of corneal endothelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells and hepatic cells.

24. The transplantation antigen-depleted cell of claim 14 wherein the transplantation antigen nucleotide sequence is a ICAM gene sequence.

25. An oligonucleotide capable of binding to a transplantation antigen nucleotide sequence through Watson-Crick binding or through triplex binding.

26. The oligonucleotide of claim 25 wherein the transplantation antigen nucleotide sequence is a MHC
class II gene sequence.

27. The oligonucleotide of claim 26 wherein the MHC class II gene sequence is located at the DR A, DP
or DQ locus.

28. The oligonucleotide of claim 27 wherein the MHC class II gene sequence is a DR A structural gene sequence or a DR A promoter sequence.

29. The oligonucleotide of claim 28 wherein the DR-A promoter sequence comprises the X-box or X2-box.

30. The oligonucleotide of claim 29 or of claim 28 wherein the oligonucleotide comprises the sequence 5' GGGGTTGGTTGTGTTGGGTGTTGTGT 3' or the sequence 5' GGTGGGGGTGGGGGTGGGGGTGGGGG 3' or fragments thereof which retain binding capability.

31. An oligonucleotide for use in the preparation of a composition for treating target cells to make them transplantation antigen-depleted, wherein the oligonucleotide is capable of binding to a portion of the transplantation antigen nucleotide sequence.

32. A universal donor organ prepared by the method comprising:
(a) obtaining a target organ from an individual; and (b) exposing the target organ to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, said oligonucleotide being preset in an amount sufficient to make the target organ a universal donor organ.

33. A method of treating an individual with an autoimmune disease characterized by dysfunctional expression of a transplantation antigen, the method 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.