AU724817B2

AU724817B2 - Method of treatment for asthma

Info

Publication number: AU724817B2
Application number: AU18574/99A
Authority: AU
Inventors: Jonathan W. Nyce
Original assignee: University of North Carolina at Chapel Hill; East Carolina University
Current assignee: East Carolina University
Priority date: 1995-06-07
Filing date: 1999-03-03
Publication date: 2000-09-28
Anticipated expiration: 2016-06-03
Also published as: AU1857499A; AU724817C

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): EAST CAROLINA UNIVERSITY Invention Title: METHOD OF TREATMENT FOR ASTHMA The following statement is a full description of this invention, including the best method of performing it-known to me/us: la Method of Treatment of Asthma The entire disclosure in the complete specification of our Australian Patent Application No.

60295/96 (699330) is by this cross-reference incorporated into the present specification.

Field of the Invention This application concerns a method of administering antisense oligonucleotides against the Ai and

A

3 Adenosine receptors as a treatment for asthma.

Description of the Background Asthma is one of the most common diseases in 15 industrialized countries, and in the United States accounts for about 1% of all health care costs. K. Weiss et al., New i Engl. J. Med. 326, 862-866 (1992). There has been reported an alarming increase in both the prevalence and mortality of asthma over the past decade, Asthma-United States, 1980- 20 1990, MMWR 41, 733-735 (1992), and occupational asthma is predicted to be the preeminent occupational lung disease in the next decade. M. Chan-Yeung and J. Malo, European Resp.

J. 7, 346-371 (1994). While the increasing mortality from asthma in industrialized countries might be attributable to 25 the increased reliance upon beta agonists in the treatment of this disease, the underlying causes of asthma remain poorly understood. J. Gern and R. Lemanske, In Immunology and Allergy Clinics of North America 13, Bush, R. K. ed.

W.B. Saunders Company, London, pp. 839-860 (1993).

Adenosine may constitute an important natural mediator of bronchial asthma. R. Pauwels et Clinical Exp. Allergy 21 Suppl. 1, 48-55 (1991); S. Holgate et al., Annals of the New York Acad. Sci. 629, 227-236 (1991). The potential role of adenosine in human asthma is supported by the experimental finding that, in contrast to normal individuals, asthmatic individuals respond to aerosolized adenosine with marked bronchoconstriction. M. Church and S.

-2 Holgate, Trends Pharmacol. Sci. 7, 49-50 (1986); M.Cushley et al., Br. J. Clin. Pharmacol. 15, 161-165 (1983).

Similarly, asthmatic rabbits produced using the dust mite allergic rabbit model of human asthma also were shown to respond to aerosolized adenosine with marked bronchoconstriction, while non asthmatic rabbits showed no response. S. Ali et al., Agents Actions 37, 165-176 (1992). Recent work using this model system has suggested that adenosine-mediated bronchoconstriction and bronchial hyperresponsiveness in asthma are mediated primarily through the stimulation of adenosine receptors. S. Ali et al., J. Pharmacol. Exp. Ther. 268, 1328-1334 (1994); S. Ali et al., Am. J. Physiol 266, L271-277 (1994).

Theophylline, an important drug in the treatment of asthma, is a known adenosine receptor antagonist (see M.

Cushley et al., Am. Rev. Resp. Dis. 129, 380-384 (1984)) and was found to eliminate adenosine-mediated bronchoconstriction in asthmatic rabbits (Ali, et al., supra). The pretreatment of allergic rabbits with another S 20 Al-specific receptor antagonist, 8-cyclopentyl-l, 3dipropylxanthine (DPCPX), potently inhibited adenosine- S: mediated bronchoconstriction and bronchial hyperresponsiveness in allergic rabbits. Id. The therapeutic potential, however, of currently available 25 adenosine Ai receptor-specific antagonists is limited by eo. their toxicity. H. Klitgaard et al., European J.

Pharmacol. 242, 221-228 (1993). Theophylline has been widely used in the treatment of asthma, but is associated with frequent, significant toxicity resulting from its .30 narrow therapeutic dose range. E. Powell et al., Paediatric Emergency Care 9, 129-133 (1993); S. Nasser and P. Rees, Drug Safety 8, 12-18 (1993); P. Epstein, Annals of Internal Med. 119, 1216-1217 (1993). The availability of an alternative strategy to downregulate adenosine-mediated bronchoconstriction would clearly be of therapeutic interest.

SIt will be clearly understood that, although a H: \Bkrot\Keep\speci\EPI-0796b.doc 26/07/00 2a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

0 6

S

.46.

s o 0 H: \Bkrot\Keep\speci\EPI-0796b.doc 26/07/00 -3 SUMMARY OF THE INVENTION The present invention provides a pharmaceutical composition, comprising an oligonucleotide (oligo), which is effective for alleviating bronchoconstriction or lung inflammation when administered to a mammal, the oligo being antisense to the initi a tion codon region, the coding region, the 5' or 3' intron-exon junctions or regions within 2 to 10 nucleotides of the junctions of a gene encoding an adenosine Al receptor or a gene encoding an adenosine A 3 receptor, or antisense to an adenosine Ai receptor mRNA or an adenosine

A

3 receptor mRNA; and a pharmaceutically acceptable carrier.

The present invention further provides a method of treating an adenosine Ai or A 3 mediated respiratory disease or condition associated with bronchoconstriction or lung inflammation, comprising administering to a subject in need of such treatment an aerosol of a pharmaceutical composition as herein described, comprising an amount of the oligo effective for alleviating bronchoconstriction and/or lung inflammation.

Also part of the present invention is a pharmaceutical composition, comprising a pharmaceutically acceptable carrier, and an adenosine receptor antisense 25 oligonucleotide. The adenosine receptor is preferably selected from the group consisting of the adenosine Ai and

A

3 adenosine receptors.

The antisense oligonucleotide of this invention may be applied to the preparation of a medicament for (a) reducing adenosine-mediated bronchoconstriction in a subject in need of such treatment, or treating asthma in a subject in need of such treatment.

Antisense oligonucleotides have received considerable theoretical consideration as potentially useful pharmacologic agents in human disease. R. Wagner, Nature.372, 333-335 (1994). However, practical applications of these molecules in actual models of human 4 disease have been elusive. One important consideration in the pharmacologic application of these molecules is route of administration. Most experiments utilizing antisense oligonucleotides in vivo have involved direct application to limited regions of the brain (see C. Wahlestedt, Trends in Pharmacological Sciences 15, 42-46 (1994); J. Lai et al., Neuroreport 5, 1049-1052 (1994); K. Standifer et al., Neuron 12, 805-810 (1994); A. Akabayashi et al., Brain Research 21, 55-61 (1994)), or to spinal fluid (see e.g. L.

Tseng et al., European J. Pharmacol. 258, R1-3 (1994); R.

Raffa et al., European J. Pharmacol. 258, R5-7 (1994); F.

Gillardon et al., European J. Neurosci. 6, 880-884 (1994)).

Such applications have limited clinical utility due to their invasive nature.

The systemic administration of antisense oligonucleotides also poses significant problems with respect to pharmacologic application, not the least of which is the difficulty in targeting disease-involved tissues. In contrast, the lung is an excellent potential 20 target for antisense oligonucleotide application since it may be approached noninvasively and in a tissue-specific manner.

BRIEF DESCRIPTION OF THE DRAWINGS 25 Figure 1 illustrates the effects of Al adenosine receptor antisense oligonucleotides and mismatch control antisense oligonucleotides on the dynamic compliance of the bronchial airway in a rabbit model. The two stars represent significant difference at p<0.01, Student's t-test.

Figure 2 illustrates the specificity of Al adenosine receptor antisense oligonucleotides as indicated by the Al and A 2 adenosine receptor number present in airway tissue treated with A 1 adenosine receptor antisense oligonucleotides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Nucleotide sequences are presented herein by 5 single strand only, in the 5' to 3' direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR 1.822 and established usage. See, PatentIn User Manual, 99-102 (Nov. 1990) Patent and Trademark Office, Office of the Assistant Commissioner for Patents, Washington, D.C.

20231); U.S. Patent No. 4,871,670 to Hudson et al. at Col.

3 lines 20-43. The relevant sections of the disclosures of this and all other patents and other references cited in this patent are incorporated herein by reference.

The method of the present invention may be used to reduce adenosine-mediated bronchoconstriction in the 15 lungs of a subject for any reason, including (but not limited to) asthma. Antisense oligonucleotides to the Ai and A 3 receptors are shown to be effective in the downregulation of Ai or A 3 in the cell. One novel feature of this treatment, as compared to traditional treatments for adenosine-mediated bronchoconstriction, is its direct administration to the lungs. The present treatment additionally selectively reduces the amount or level of a receptor protein itself rather than as is the case with treatments wehre the agent merely interacts with the 25 receptor. The selective characteristic of the present antisense oligonucleotide results in a reduction in toxicity.

As used herein, the term "treat" or "treating" asthma refers to a treatment which decreases the likelihood that the subject administered such treatment will manifest symptoms of bronchoconstriction or asthma. The term "downregulate", thus, refers to inducing a decrease in production, secretion or availability, and thus a decrease in concentration, of intracellular Ai or A 3 adenosine receptor.

The present invention is concerned primarily with the treatment of human subjects but may also be employed 6 for the treatment of other mammalian subjects, such as dogs and cats, for veterinary purposes.

In general, "antisense" refers to the use of small, synthetic oligonucleotides, resembling singlestranded DNA, to inhibit gene expression by inhibiting the function of the target messenger RNA (mRNA). Milligan, J.

F. et al., J. Med. Chem. 36(14), 1923-1937 (1993). The present invention, thus, is intended for inhibition of gene expression of the Ai or A 3 adenosine receptor. As is generally known, gene expression may be inhibited through oligonucleotide hybridization to coding (sense) sequences in a specific messenger RNA (mRNA) target, by hydrogen bonding according to Watson-Crick base pairing rules. In general, the exogenously applied oligonucleotides 15 decrease the mRNA and protein levels of the target gene or cause changes in the growth characteristics or shapes of the cells. Id. See also Helene, C. and Toulme, Biochim.

Biophys. Acta 1049, 99-125 (1990); Cohen, J. Ed., Oligodeoxynucleotides as Antisense Inhibitors of Gene 20 Expression; CRC Press: Boca Raton, FL (1987).

As used herein, "adenosine receptor antisense oligonucleotide" is defined as a short sequence of synthetic nucleotides that hybridizes to any coding sequence in an mRNA which codes for an adenosine receptor, 25 the Ai adenosine receptor or A 3 adenosine receptor, according to hybridization conditions described below, and upon hybridization causes a decrease in gene expression of the Ai or A 3 adenosine receptor.

The mRNA sequence of the Ai or A 3 adenosine receptor may be derived from the DNA base sequence of the gene expressing either the Ai or A 3 adenosine receptor.

The sequence of the genomic human Ai adenosine receptor is known and is disclosed in U.S. Patent No. 5,320,962 to G.

Stiles et al. The A 3 adenosine receptor has been cloned, sequenced and expressed in rat (see F. Zhou et al., Proc.

Nat'l Acad. Sci. USA 89 :7432 (1992)) and humans (see M.A.

Jacobson et al., U.K. Patent Application No. 9304582.1 7 (1993)). The antisense oligonucleotides that downregulate the production of the Ai or A 3 adenosine receptor may be produced in accordance with standard techniques.

The antisense oligonucleotide of this invention binds specifically with any sequence of an mRNA molecule which encodes a human receptor and prevents one embodiment of the oligonucleotide has a SEQ ID NO: 3, and SEQ -GAT GGA -GTT GTT -GTG GGC In another embodiment Ai adenosine receptor or A 3 -adenosine translation of the mRNA molecule. In present invention, the antisense sequence identified as SEQ ID NO: 1, ID NO: 5, shown below.

GGG CGG CAT GGC GGG-3' (SEQ ID NO:1) GGG CAT CTT GCC-3' (SEQ ID NO:3) CTA GCT CTC GCC-3' (SEQ ID of the invention, the sequence of the *9 antisense oligonucleotide brackets the initiation codon of the human adenosine Ai receptor. Such an antisense oligonucleotide follows: GGC CTG GAA GGC-3' 5'-GGC GGC CTG GAA 3' may have a sequence disclosed herein as AGC TGA GAT GGA AGC TGA GAT GGA 3' GGC CTG GAA AGC TGA GAT GGA 5'-GGC GGC CTG GAA AGC TGA GAT GGA GGC CTG GAA AGC TGA GAT GGA GGC CTG GAA AGC TGA GAT GGA GGC CTG GAA AGC TGA GAT GGA GGC CTG GAA AGC TGA GAT GGA 5'-GGC GGC CTG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG (SEQ. ID NO:7) GGG CGG CAT GGC GGG (Fragment 1) (SEQ.

GGG CGG CAT GGC GGG (Fragment 2) (SEQ.

GGG CGG CAT GGC GGG (Fragment 3) (SEQ.

GGG CGG CAT GGC GGG (Fragment 4) (SEQ.

GGG CGG CAT GGC GGG (Fragment 5) (SEQ.

GGG CGG CAT GGC GGG (Fragment 6) (SEQ.

GGG CGG CAT GGC GGG (Fragment 7) (SEQ.

GGG CGG CAT GGC GGG (Fragment 8) (SEQ.

CAC AGG CTG CAC AGG CTG GG- ID NO:8) CAC AGG CTG G- ID NO:9) CAC AGG CTG -3' ID CAC AGG CT-3' ID NO:11) CAC AGG C-3' ID NO:12) CAC AGG -3' ID NO:13) CAC AG-3' ID NO:14) CAC A-3' ID GGC CTG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC-3' -8

-GGC

5'-GGC

-GGC

5' -GGC 15 5' -GGC

-GGC

5'-GGC 5' -GGC

-GGC

5'-GGC

-GGC

CTG

GAA

AGC

TGA

GAT

GGA

(Fragment 9) (SEQ. ID NO:16) GGG CGG CAT GGC GGG CA-31 (Fragment 10) (SEQ. ID NO:17) GOG CGG CAT GGC GGG C-31 (Fragment 11) (SEQ. ID NO:18) GGG CGG CAT GGC GGG -3' (Fragment 12) (SEQ. ID NO:19) GGG CGG CAT GGC GG-3' (Fragment 13) (SEQ. ID GGG, CGG CAT GGC G-3' (Fragment 14) (SEQ. ID NO:21) GGG CGG CAT GGC -3' (Fragment 15) (SEQ. ID NO:22) GGG CGG CAT GG -3' (Fragment 16) (SEQ. ID NO:23) GGG, CGG CAT G -3' (Fragment 17) GGG CGG CAT 3 (Fragment 18) GGG CGG CA-31 (Fragment 19) GGG CGG C-31 (Fragment 20) GGG CGG -3' (Fragment 21) GGG CG 3 (Fragment 22) GGG C -3' (Fragment 23) GGG -3' (Fragment 24) OG -3' (Fragment 25) G -3' (Fragment 26) -31 (Fragment 27) (SEQ. ID NO:24)

(SEQ.

NO: NO: 2 6) NO: 27) NO 2 8) NO: 2 9) NO: 3 0) NO: 31) NO: 3 2) NO 3 3) NO: 3 4) 9-

-GGC

'-GOC

S '-GOC

'-GGC

5'-GGC

'-GGC

5'-GGC

'-GGC

-OGC

5 '-GGC 15 5'-GCC 3,' 3,'

GGC

GC

GGC

GC

GGC

C

GGC

GC

GAA

AGC TGA OAT GO -3' AGC TGA GAT G -3' AGC TGA GAT -3' AGC TGA GA-3' AGC TGA G-3' AGC TGA-3' AGC TG-3' AGC T-3 AGC-3' AG-3' A-3' GAA-3' GA-3' G-3' Sn.

'S S S 'GC CTO OAA AGC TGA GAT GGA 000 3GC CTG GAA AGC TGA GAT GGA GGG (Fragment 28) (Fragment 29) (Fragment 30) (Fragment 31) (Fragment 32) (Fragment 33) (Fragment 34) Fragment 35) (Fragment 36) (Fragment 37) (Fragment 38) (Fragment 39) (Fragment 40) (Fragment 41) CGG CAT GOC GO (Fragment 42) CGG CAT GOC GO (Fragment 43) COG CAT GGC 000 (Fragment 44) CGO CAT GGC 000 (Fragment 45) CGO CAT GGC 000 (Fragment 46) CGG CAT GGC GGO (Fragment 47) COG CAT GGC GG (Fragment 48) CGG CAT GOC 000 (Fragment 49) COG CAT GOC 000 (Fragment 50) COG CAT GOC 000 (Fragment 51) COG CAT GOC 000 (Fragment 52) SEQ. ID SEQ. ID NO:36) SEQ. ID NO:37) (SEQ. ID NO:38) (SEQ. ID NO:39) (SEQ. ID (SEQ. ID NO:41) (SEQ. ID NO:42) (SEQ. ID NO:43) (SEQ. ID NO:44) (SEQ. ID (SEQ. ID NO:46) (SEQ. ID NO:47) (SEQ. ID NO:48) CAC AGO CTG GOC- (SEQ. ID NO:49) CAC AGO CTG 00- (SEQ. ID CAC AGO CTG G-3' (SEQ. ID NO:51) CAC AGO CTG -3' (SEQ. ID NO:52) CAC AGO CT-3' (SEQ. ID NO:53) CAC AGO C-3' (SEQ. ID NO:54) CAC AGO -3' (SEQ. ID CAC AG-3' (SEQ. ID NO:56) CAC A-3' (SEQ. ID NO:57) CAC-3' (SEQ. ID NO:58) CA-3' (SEQ. ID NO:59) 5'-GC GOC CTG GAA AGC TOA OAT OGA 000 GOC CTG GAA AGC TGA OAT OGA 000 5'-GC GOC CTO GAA AGC TGA OAT OGA GOG 25 5'-GC GOC CTG GAA AGC TGA OAT OGA 000 GOC CTO GAA AOC TGA OAT OGA GG GOC CTC GAA AOC TGA OAT GOA GO GOC CTG OAA AGC TOA OAT OCA 000 GOC CTO OAA AGC TGA OAT GOAGOG 5'-OC GOC CTO OAA AOC TCA OAT OGA 000 GOC CTO OAA ACC TGA OAT OGA 000 COG CAT 0CC GOG C-3' 10

-GC

'-GC

-GC

5'-GC

'-GC

15 a.

~5'-GC 5'-GC 20 5'-GC 0.

-GC

5' -GC

-GC

5'-GC

-GC

'-GC

5'-GC

CTG

CTG,

CTG

CTG,

CTG

CTG,

CTG

AGC

TGA

GAT

GCA GGG GGA GGG GGA GGG GGA CG GCA GGG GGA GGG GGA GGG GCA GGG GGA GGG GGA GGG GGA GGG GGA CG GGA GGG GGA GG GGA C GGA -3' GG -3' G -3' (Fragment 53) (SEQ.

CGG CAT GGC GGG -3' (Fragment 54) (SEQ.

CGG CAT GGC GG-3' (Fragment 55) (SEQ.

CGG CAT GGC G-31 NO: 6 0) NO: 6 1) NO: 62) (Fragment 56) CGG CAT GGC -3' (Fragment 57) CGG CAT GG -3' (Fragment 58) CGG CAT G -3' (Fragment 59) COG CAT -3' (Fragment 60) CGG CA-31 (Fragment 61) CGG C-3' (Fragment 62) CGG -3' (Fragment 63) CG -3' (Fragment 64) C -3' (Fragment 65) -3, (Fragment 66) -3, (Fragment 67) -3, (Fragment 68) (Fragment 69) (Fragment 70) (Fragment 71) (SEQ. ID NO:63)

(SEQ.

NO: 64) NO: NO: 66) NO: 67) NO: 6 8) NO: 6 9) NO: 7 0) NO: 7 1) NO: 7 2) NO: 73) NO: 74) NO: 7 NO: 7 6) NO :77) NO: 7 8) GGC CTG GAA AGC TGA GAT -3' GGC CTG GAA AGC TGA'GA-3' (Fragment 72) (SEQ. ID NO:79) (Fragment 73) (SEQ. ID 11 S' -GC

-GC

'-GC

'-C

5'-GC

'-C

'-GC

S '-C

'-C

5,-Cc 3,

GAA

ACC TGA G-3' AGC TGA-3' ACC TG-3' AGC T-3' CAA AGC-3' CAA AG-V' GAA A-3' GAA-3' GA-3' GC CTG GAA AGC TGA GAT GGA GGG see* :05005 *000.

44060 GGC CTG GAA AGC TGA GAT GGA GG CCC CTC GAA AGC TCA GAT GGA CCC CCC CTG GAA AGC TGA CAT CCA CG 5'-C GGC CTC CAA AGC TGA GAT CGA GGG 20 5'-C CGC CTG CAA AGC TGA GAT CGA GGG CGC CTG GAA AGC TGA GAT CGA GGG GGC CTG CAA AGC TGA GAT GGA GC GGC CTG GAA AGC TGA GAT CGA GGG GGC CTG GAA AGC TGA GAT GCA GG 5'-C GGC CTG CAA AGC TGA GAT GCA GGG GGC CTG GAA AGC TGA GAT GGA GGG GGC CTG CAA AGC TGA GAT CCA GGG CCC CTG CAA ACC TGA GAT CGA GGG (Fragment 74) (Fragment 75) (Fragment 76) (Fragment 77) (Fragment 78) (Fragment 79) (Fragment 80) (Fragment 81) (Fragment 82) CGG CAT CCC CCC (Fragment 83) CCC CAT CCC CCC (Fragment 84) CCC CAT CCC CCC (Fragment 85) CCC CAT CCC CCC (Fragment 86) CCC CAT CCC CCC (Fragment 87) CCC CAT CCC CCC (Fragment 88) CCC CAT CCC CCC (Fragment 89) CCC CAT CCC CCC (Fragment 90) CGC CAT CCC CCC (Fragment 91) CCC CAT CCC CCC (Fragment 92) CCC CAT CCC CCC (Fragment 93) CCC CAT CCC CCC (SEQ. ID NO:81) (SEQ. ID NO:82) (SEQ. ID NO:83) (SEQ. ID NO:84) (SEQ. ID (SEQ. ID NO:86) (SEQ. ID NO:87) (SEQ. ID NO:88) (SEQ. ID NO:89) CAC ACC CTC CCC- (SEQ. ID CAC ACC CTC CC-V' (SEQ. ID NO:91) CAC ACC CTC G-3' (SEQ. ID NO:92) CAC ACC CTC -3' (SEQ. ID NO:93) CAC ACC CT-3' (SEQ. ID NO:94) CAC ACC C-3' (SEQ. ID CAC ACC -3' (SEQ. ID NO:96) CAC AC-3' (SEQ. ID NO:97) CAC A-3' (SEQ. ID NO:98) CAC-3' (SEQ. ID NO:99) CA-3' SEQ. ID NO:100) C-3' (Fragment 94) (SEQ.

CCC CAT CCC CCC -3' (Fragment 95) (SEQ.

CCC CAT CCC GG-3' ID NO:101) ID NO: 102) (Fragment 96) (SEQ. ID NO:103) oo Sees o e

C

5 C S 5

S

C

0

C

m ooooo ovid °oo 5'-C 5'-C 15 5'-C 5'-C 5'-C 20 5'-C 25 5'-C -c 5'-C 5'-C 5'-C

GGC

CTG

GAA

AGC

12 TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GGG TGA GAT GGA GG TGA GAT GGA G TGA GAT GGA -3' TGA GAT GG -3' TGA GAT G -3' TGA GAT -3' TGA GA-3' TGA G-3' TGA-3' TG-3' T-3' n CGG CAT GGC G-3' (Fragment 97) CGG CAT GGC -3' (Fragment 98) CGG CAT GG -3' (Fragment 99) CGG CAT G -3' (Fragment 100) CGG CAT -3' (Fragment 101) CGG CA-3' (Fragment 102) CGG C-3' (Fragment 103) CGG -3' (Fragment 104) CG -3' (Fragment 105) C -3' (Fragment 106) -3' (Fragment 107) -3' (Fragment 108) -3' (Fragment 109) (Fragment 110) (Fragment 111) (Fragment 112) (Fragment 113) (Fragment 114) (Fragment 115) (Fragment 116) (Fragment 117) (Fragment 118)

(SEQ.

ID NO:104) ID NO:105) ID NO:106) ID NO:107) ID NO:108) ID NO:109) ID NO:110) ID NO:111) ID NO:112) ID NO:113) ID NO:114) ID NO:115) ID NO:116) ID NO:117) ID NO:118) ID NO:119) ID NO:120) ID NO:121) ID NO:122) ID NO:123) ID NO:124) ID NO:125) GGC CTG GAA AGC-3' GGC CTG GAA AG-3' (Fragment (Fragment 119) 120)

(SEQ.

NO:126) NO:127) 13 GGC CTG GAA A-3' GGC CTG GAA-3' (Fragment 121) (SEQ. ID NO:128) (Fragment 122) (SEQ. ID NO:129) GGC CTG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' .0.

:0.0*0 5'- 51- 5'- 25 5'- 5,-

GGC

CTG

CTG,

CTG

GAA

AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA

GAT

GGA GGG GGA GGG GGA GGG GGA GGG GGA GGG GGA GGG GGA GGG GdA GGG GGA GGG GGA GGG GGA GG GGA GGG GGA GGG GGA GGG GGA GGG GGA GGG (Fragment 123) (SEQ. ID NO:130) CGG CAT GGC GGG CAC AGG CTG GG-3' (Fragment 124) (SEQ. ID NO:131) CGG CAT GGC GGG CAC AGG CTG G-3' (Fragment 125) (SEQ. ID NO:132) CGG CAT GGC GGG CAC AGG CTG -3' (Fragment 126) (SEQ. ID NO:133) CGG CAT GGC GGG CAC AGG CT-V' (Fragment 127) (SEQ. ID NO:134) CGG CAT GGC GGG CAC AGG C-31 (Fragment 128) (SEQ. ID NO:135) CGG CAT GGC GGG CAC AGG -3' (Fragment 129) (SEQ. ID NO:136) CGG CAT GGC GGG CAC AG-V1 (Fragment 130) (SEQ. ID NO:137) CGG CAT GOC GGG CAC A-3' (Fragment 131) (SEQ. ID NO:138) CGG CAT GGC GGG CAC-3' (Fragment 132) (SEQ. ID NO:139) CGG CAT GGC GGG CA-3' (Fragment 133) (SEQ. ID NO:140) CGG CAT GGC GGG C -3 (Fragment 134) (SEQ. ID NO:141) CGG CAT GGC GGG -3' (Fragment 135) (SEQ. ID NO:142) CGG CAT GGC GG-3' (Fragment 136) (SEQ. ID NO:143) CGG CAT GGC G-3' (Fragment 137) (SEQ. ID NO:144) CGG CAT GGC -3' (Fragment 138) (SEQ. ID NO:145) CGG CAT GG -3' (Fragment 139) GGC CTG GAA AGC TGA GAT GGA GGG CGG CAT G -3' (SEQ. ID NQ:146) 14 GGC CTG GAA AGC TGA GAT GGA GGG GGC CTG GAA AGC TGA GAT GGA GGG GGC CTG GAA AGC TGA GAT GGA GGG GGC CTG GAA AGC TGA GAT GGA GGG (Fragment 140) (SEQ. ID NO:147) CGG CAT -3' (Fragment 141) (SEQ. ID NO:148) CGG CA-31 (Fragment 142) (SEQ. ID NO:149) CGG C-31 (Fragment 143) (SEQ. ID NO:150) CGG -3' (Fragment 144) GGC CTG GAA AGC TGA GAT GGA GGG CG -3' (Fragment 145) GGC CTG GAA AGC TGA GAT GGA GGG C -3' (SEQ. ID NO:151) (SEQ. ID NO:152) (SEQ. ID NO:153) (SEQ. ID NO:154) (Fragment 146) GGC CTG GAA AGC TGA GAT GGA GGG -3' (Fragment 147) GGC CTG GAA AGC TGA GAT GGA GG -3' 5'- 5'- 5'- 5'- 5'- 5'-

GGC

CTG

GAA

AGC TGA GAT GGA G -3' AGC TGA GAT GGA -3' AG C TGA GAT GG -3' AGC TGA GAT G -3' AGC TGA GAT -3' AGC TGA GA-3' AGC TGA G-3' AGC TGA-3' AGC TG-3' AGC T-3' AGC-3' AG-3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment Fragment (Fragment (Fragment (Fragment (Fragment 148) 149) 150) 151) 152) 153) 154) 155) 156) 157) 158) 159) 160) 161)

(SEQ.

AGG

(SEQ.

AGG

ID

CTG

ID

CTG

NO: 155) NO: 156) NO: 157) NO: 158) NO: 159) NO: 160) NO: 161) NO: 162) NO: 163) NO: 164) NO: 165) NO: 166) NO: 167) NO: 168) GGC-3' NO: 169) GG-3' NO: 17 0) G-3' NO: 171) -3' GC CTG GAA AGC TGA GAT GGA GGG CGG GC CTG GAA AGC TGA GAT GGA GGG CGG GC CTG GAA AGC TGA GAT GGA GGG CGG GC CTG, GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC (Fragment 162) CAT GGC GGG CAC (Fragment 163) (SEQ. ID CAT GGC GGG CAC AGG CTG (Fragment 164) (SEQ. ID CAT GGC GGG CAC AGG CTG is (Fragment 165) (SEQ. ID NO:172) GC CTG GAA AGC TGA GAT GGA GGG CGG CAT GOC GGG CAC AGG CT-3' (Fragment 166) (SEQ. ID NO:173) GC CTG GAA AGC TGA GAT GGA GGG CGG CAT GGC GO CAC AGG C-3' (Fragment 167) (SEQ. ID NO:174) GC CTG GAA AGC TGA GAT GGA COG CGG CAT GGC GOG CAC AGG -3' (Fragment 168) (SEQ. ID NO:175) GC CTG GAA AGC TGA GAT GGA GOG CGG CAT GGC GOC CAC AG-3' (Fragment 169) (SEQ. ID NO:176) GC CTG GAA AGC TGA GAT GGA GGG CGG CAT GGC 000 CAC A-3' (Fragment 170) (SEQ. ID NO:177) GC CTG GAA AGC TGA GAT GGA GGG COG CAT 0CC COG CAC-3' (Fragment 171) (SEQ. ID NO:178) GC CTO GAA AGC TGA OAT OGA GGG CGG CAT 0CC GGG CA-3' 15 (Fragment 172) (SEQ. ID NO:179) GC CTG OAA AGC TGA GAT GGA G CGG CAT GGC COO C-3' (Fragment 173) (SEQ. ID NO:180) 0 GC CTG GAA AGC TGA OAT GGA GGG CGG CAT GOC GGG -3' (Fragment 174) (SEQ. ID NO:181) GC CTG GAA AGC TGA CAT GGA GGG COG CAT CCC GG-3' (Fragment 175) (SEQ. ID NO:182) GC CTG GAA AGC TGA GAT OCA COG CGG CAT GGC G-3' (Fragment 176) (SEQ. ID NO:183) GC CTG GAA AGC TCA GAT GCA COG CGG CAT GGC -3' (Fragment 177) (SEQ. ID NO:184) GC CTG GAA AGC TGA CAT GGA GGG CGG CAT GG -3' 0 (Fragment 178) (SEQ. ID NO:185) GC CTG GAA AGC TGA OAT GCA GGG COG CAT G -3' (Fragment 179) (SEQ. ID NO:186) GC CTG GAA ACC TGA CAT GGA GGG CGG CAT -3' (Fragment 180) (SEQ. ID NO:187) GC CTC OAA ACC TGA OAT OGA GGG CGG CA-3' (Fragment 181) (SEQ. ID NO:188) GC CTG OAA ACC TGA GAT OGA CGG COG C-3' (Fragment 182) (SEQ. ID NO:189) GC CTG CAA AGC TGA GAT GGA GOG COG -3' (Fragment 183) (SEQ. ID NO:190) 16 GC CTG GAA AGC TGA GAT GGA GGG CG -3' (Fragment 184) (SEQ. ID NO:191) GC CTG GAA AGC TGA GAT GGA GGG C -3' GC CTG GAA AGC TGA GAT GGA GGG -3' GC CTG GAA AGC TGA GAT GGA GG -3' (Fragment 185) (SEQ. ID NO:192) (Fragment 186) (SEQ. ID NO:193) 5'- 51- 5'- 5'- 5'- 5'- GAA AGC TGA GAT GGA G -3' GAA AGC TGA GAT GGA -3' GAA AGC TGA GAT GG -3' GAA AGC TGA GAT G -3' GAA AGC TGA GAT -3' GAA AGC TGA GA-3' GAA AGC TGA G-3' GAA AGC TGA-3' GAA AGC TG-3' GAA AGC T-3' GAA AGC-3' GAA AG-3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 187) 188) 189) 190) 191) 192) 193) 194) 195) 196) 197) 198) C CTG GAA AGC TGA GAT GGA GGG CGG C CTG GAA AGC TGA GAT GGA GGG CGG C CTG GAA AGC TGA GAT GGA GGG CGG C CTG GAA AGC TGA GAT GGA GGG CGG C CTG GAA AGC TGA GAT GGA GGG CGG C CTG GAA AGC TGA GAT GGA GGG CGG C CTG GAA AGC TGA GAT GGA GGG CGG C CTG GAA AGC TGA GAT GGA GGG CGG (Fragment 199) CAT GGC GGG CAC (Fragment 200) CAT GGC GGG CAC (Fragment 201) CAT GGC GGG CAC (Fragment 202) CAT GGC GGG CAC (Fragment 203) CAT GGC GGG CAC (Fragment 204) CAT GGC GGG CAC (Fragment 205) CAT GGC GGG CAC (Fragment 206) CAT GGC GGG CAC (Fragment 207) (SEQ. ID NO:194) (SEQ. ID NO:195) (SEQ. ID NO:196) (SEQ. ID NO:197) (SEQ. ID NO:198) (SEQ. ID NO:199) (SEQ. ID NO:200) (SEQ. ID NO:201) (SEQ. ID NO:202) (SEQ. ID NO:203) (SEQ. ID NO:204) (SEQ. ID NO:205) (SEQ. ID NO:206) AGG CTG GGC-3' (SEQ. ID NO:207) AGG CTG GG-3' (SEQ. ID NO:208) AGG CTG G-3' (SEQ. ID NO:209) AGG CTG -3' (SEQ. ID NO:210) AGG CT-3' (SEQ. ID NO:211) AGG C-3' (SEQ. ID NO:212) AGG -3' (SEQ. ID NO:213) AG-3' (SEQ. ID NO:214) C CTG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC A-3' 17 (Fragment 208) (SEQ.

5'- 15 5'- 5 5'- 5'- 5'-

CTG

GAA

AGC

TGA

GAT

GGA

GGG CGG CAT GGC GGG CAC- (Fragment 209) GGG CGG CAT GGC GGG CA-3 (Fragment 210) GGG CGG CAT GGC GGG C-3' (Fragment 211) GGG CGG CAT GGC GGG -3' (Fragment 212) GGG CGG CAT GGC GG-3' (Fragment 213) GGG CGG CAT GGC G-3' (Fragment 214) GGG CGG CAT GGC -3' (Fragment 215) GGG CGG CAT GG -3' (Fragment 216) GGG CGG CAT G -3' (Fragment 217) GGG CGG CAT -3' (Fragment 218) GGG CGG CA-3' (Fragment 219) GGG CGG C-3' (Fragment 220) GGG CGG -3' (Fragment 221) GGG CG -3' (Fragment 222) GGG C -3' (Fragment 223) GGG (Fragment 224) GG (Fragment 225) G (Fragment 226) (Fragment 227) 3'

(SEQ.

I

NO:215) NO:216) (SEQ. ID NO:217)

(SEQ.

NO:218) NO:219) NO:220) NO:221) NO:222) NO:223) NO:224) NO:225) N0:226) NO:227) NO:228) N0:229) NO:230) NO:231) NO:232) NO:233) NO:234) NO:235) NO:236)

C

CTG GAA AGC TGA GAT GG -3' CTG GAA AGC TGA GAT G -3' (Fragment (Fragment 228) 229) 18 C CTG GAA AGC TGA GAT -3' C CTG GAA AGC TGA GA-3' C CTG GAA AGC TGA G-3' C CTG GAA AGC TGA-3' C CTG GAA AGC TG-3' C CTG GAA AGC T-3' C CTG GAA AGC-3' CTG GAA AGC TGA

S

a 4*

S

S S

S

*5*S 5'- 5'- 5'- 5'- 5'- 5'- 5'- 5'-

CTG

AGC

TGA

GAT

GGA

GGG

CGG

CGG.

CGG

(Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG (Fragment CAT GGC GGG 230) (SEQ. ID NO:237) 231) (SEQ. ID NO:238) 232) (SEQ. ID NO:239) 233) (SEQ. ID NO:240) 234) (SEQ.. ID NO:241) 235) (SEQ. ID NO:242) 236) (SEQ. ID NO:243) CAC'AGG CTG GGC-3' 237) (SEQ. ID NO:244) CAC AGG CTG GG-3' 238) (SEQ. ID NO:245) CAC AGG CTG G-3' 239) (SEQ. ID NO:246) CAC AGG CTG -3' 240) (SEQ. ID NO:247) CAC AGG CT-3' 241) (SEQ. ID NO:248) CAC AGG C-3' 242) (SEQ. ID NO:249) CAC AGG -3' 243) (SEQ. ID NO:250) CAC AG-3' 244) (SEQ. ID NO:251) CAC A-3' 245) (SEQ. ID NO:252) CAC-3' 246) (SEQ. ID NO:253) CA-3' 247) (SEQ. ID NO:254) C-3' 248) (SEQ. ID NO:255) -3' (Fragment 249) CAT GGC GG-3' (Fragment 250) (SEQ. ID NO:256) (SEQ. ID NO:257) CTG GAA AGC TGA GAT GGA GGG CGG CAT GGC G-3' (Fragment 251) (SEQ. ID NO:258) 19 CTC GAA AGC TGA GAT CCA GC CTG CAA AGC TCA GAT CCA GG CTG CAA AGC TGA GAT GGA GG CTC GAA AGC TGA CAT GCA GG CTG GAA AGC TGA GAT GGA GGG CCG CAT GCC -3' (Fragment CCG CAT GC -3' (Fragment CCG CAT G -3' (Fragment CCG CAT -3' (Fragment CGG CA-3' (Fragment 252) (SEQ. ID NO:259) 253) (SEQ. ID NO:260) 254) (SEQ. ID NO:261) 255) (SEQ. ID NO:262) 256) (SEQ. ID NO:263) CTG GAA AGC TGA CAT CCA GGG CGG C-3' (Fragment 257) (SEQ. ID NO:264) CTG CAA AGC TGA CAT GGA GGG CGG -3' (Fragment 258) (SEQ. ID NO:265) CTG GAA AGC TCA GAT CCA CCCG CC -3' 9 69 5'- 5'- 5'- 5'- 25 5'- 5'-

CTC

CTG

CTC

CAA

TGA CAT CCA CGG C -3' TCA CAT CCA GCG -3' TGA CAT CCA CC -3' TCA CAT CCA C -3' TGA CAT CCA -3' TGA CAT CC -3' TCA CAT C -3' TGA CAT -3' TCA CA-3' TCA C-3' TCA-3' TC-3' T-3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 259) 260) 261) 262) 263) 264) 265) 266) 267) 268) 269) 270) 271) 272)

(SEQ.

NO: 266) NO :267) NO: 268) NO: 269) NO: 270) NO 271) NO: 272) NO 273) NO: 274) NO: 275) NO :276) NO: 277) NO 278) NO: 279) *Do TC GAA ACC TCA CAT CCA CCC CCC TC CAA ACC TCA CAT CCA CCC CCC TC CAA ACC TCA CAT CCA GC CCC TG CAA ACC TCA CAT CCA CCC CCC CAT GGC GGG CAC ACC CTC (Fragment 273) (SEQ.

CAT GGC GGG CAC ACC CTC (Fragment 274) (SEQ.

CAT GGC GGG CAC ACC CTC (Fragment 275) (SEQ.

CAT GGC GGG CAC ACC CTG (Fragment 276) (SEQ.

GCC-3' ID NO:280) GC-3' ID NO:281) G-3' ID NO:282) -3' ID NO:283) 20 TG GAA AGC TGA GAT GGA GGG CGG CAT GOC GOG CAC AGG CT-3' (Fragment 277) (SEQ. ID NO:284) TG GAA AGC TGA GAT OGA GGG CGG CAT GGC GGG CAC AGG C-3' (Fragment 278) (SEQ. ID NO:285) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG -3' (Fragment 279) (SEQ. ID NO:286) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AG-3' (Fragment 280) (SEQ. ID NO:287) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC A-31 (Fragment 281) (SEQ. ID NO:288) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC-31 (Fragment 282) (SEQ. ID NO:289) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CA-31 :(Fragment 283) (SEQ. ID NO:290) *.15 TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG C-31 (Fragment 284) (SEQ. ID NO:291) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG -3' (Fragment 285) (SEQ. ID NO:292) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC GG-3' (Fragment 286) (SEQ. ID NO:293) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC G-31 (Fragment 287) (SEQ. ID NO:294) TG GAA AGC TGA GAT GGA GGG CGG CAT GGC -3' *(Fragment 288) (SEQ. ID NO:295) TG GAA AGC TGA GAT GGA GGG CGG CAT GG -3' (Fragment 289) (SEQ. ID NO:296) TG GAA AGC TGA GAT GGA GGG CGG CAT G -3' (Fragment 290) (SEQ. ID NO:297) TG GAA AGC TGA GAT GGA GGG CGG CAT -3' (Fragment 291) (SEQ. ID NO:298) TG GAA AGC TGA GAT GGA GGG CGG CA-3' (Fragment 292) (SEQ. ID NO:299) TG GAA AGC TGA GAT GGA GGG CGG C-3' (Fragment 293) (SEQ. ID NO:300) TG GAA AGC TGA GAT GGA GGG CGG -31 (Fragment 294) (SEQ. ID NO:301) TG GAA AGC TGA GAT GGA GGG CG -3' 21 5'-

GAA

AGC

GGA GGG C -3 GGA GGG -3' GGA GG -3' GGA G -3' GGA -3' GG -3' G -3' -3' TG GAA AGC TGA GA-3' TG GAA AGC TGA G-3' TG GAA AGC TGA-3' TG GAA AGC TG-3' G GAA AGC TGA GAT GGA GGG CGG CAT G -GAA AGC TGA GAT GGA GGG CGG CAT (Fragment 2 95) (Fragment 296) (Fragment 297) (Fragment 298) (Fragment 299) (Fragment 300) (Fragment 301) (Fragment 302) (Fragment 303) (Fragment 304) (Fragment 305) (Fragment 306) (Fragment 307) GGC GGG CAC AGG (Fragment 308) GGC GGG CAC AGG (Fragment 309) GGC GGG CAC AGG (Fragment 310) GGC GGG CAC AGG (SEQ. ID NO:302) (SEQ. ID NO:303) (SEQ. ID NO:304) (SEQ. ID NO:305) (SEQ. ID NO:306) (SEQ. ID NO:307) (SEQ. ID NO:308) (SEQ. ID NO:309) (SEQ. ID NO:310) (SEQ. ID NO:311) (SEQ. ID NO:312) (SEQ. ID NO:313) (SEQ. ID NO:314) CTG GGC-3' (SEQ. ID NO:315) CTG GG-3' (SEQ. ID NO:316) CTG G-3' (SEQ. ID NO:317) CTG -3' G GAA AGC TGA GAT GGA GGG CGG CAT G GAA AGC TGA GAT GGA GGG CGG CAT G GAA AGC TGA GAT GGA GGG CGG CAT G GAA AGC TGA GAT GGA GGG CGG CAT G GAA AGC TGA GAT GGA GGG CGG CAT 59'- G GAA AGC TGA GAT GGA GGG CGG CAT (Fragment 311) (SEQ. ID NO:318) GGC GGG CAC AGG CT-3' (Fragment 312) (SEQ. ID NO:319) GGC GGG CAC AGG C-3' (Fragment 313) (SEQ. ID NO:320) GGC GGG CAC AGG -3' (Fragment 314) (SEQ. ID NO:321) GGC GGG CAC AG-3' (Fragment 315) (SEQ. ID NO:322) GGC GGG CAC A-3' (Fragment 316) (SEQ. ID NO:323) GGC GGG CAC-3' (Fragment 317) (SEQ. ID NO:324) GGC GGG CA-31 (Fragment 318) (SEQ. ID NO:325) GGC GGG C-3' (Fragment 319) (SEQ. ID NO:326) G GAA AGC TGA GAT GGA GGG CGG CAT G'GA.A AGC TGA GAT GGA GGG CGG CAT G GAA AGC TGA GAT GGA GGG CGG CAT G GAA AGC TGA GAT GGA GGG CGG CAT 22 5'- 15 5'- 5, 5,- 5, 5'- 5'-

GAA

AGC

GAT GGA GGG CGG CAT GGC GGG -3' (Fragment 320) GAT GGA GGG CGG CAT GGC GG -3' (Fragment 321) GAT GGA GGG CGG CAT GGC G-3' (Fragment 322) GAT GGA GGG CGG CAT GGC -3 (Fragment 323) GAT GGA GGG CGG CAT OG -3' (Fragment 324) GAT GGA GGG CGG CAT G -3' (Fragment 325) GAT GGA GGG CGG CAT -3' (Fragment 326) GAT GGA GGG CGG CA-3' (Fragment 327) GAT GGA GGG CGG C-3' (Fragment 328) GAT GGA GGG CGG -3' (Fragment 329) GAT GGA GGG CG (Fragment 330) GAT GGA GGG C -31. (Fragment 331) GAT GGA GGG (Fragment 332) GAT GGA GG (Fragment 333) GAT GGA G (Fragment 334) GAT GGA (Fragment 335) GAT GG (Fragment 336) GAT G (Fragment 337) GAT (Fragment 338) GA-3' (Fragment 339) G-3' (Fragment 340) (SEQ. ID NO:327) (SEQ. ID NO:328) (SEQ. ID NO:329) (SEQ. ID NO:330) (SEQ. ID NO:331) (SEQ. ID NO:332) (SEQ. ID NO:333) (SEQ. ID NO:334) (SEQ. ID NO:335) (SEQ. ID NO:336) (SEQ. ID NO:337) (SEQ. ID NO:338) (SEQ. ID NO:339) (SEQ. ID NO:340) (SEQ. ID NO:341) (SEQ. ID NO:342) (SEQ. ID NO:343) (SEQ. ID NO:344) (SEQ. ID NO:345) (SEQ. ID NO:346) (SEQ. ID NO:347) (SEQ. ID NO:348) CTG GGC-3' (SEQ. ID NO:349) CTG GG-3' (SEQ. ID NO:350) CTG G-3' G GAA AGC TGA-3' GAA AGC TGA GAT GGA GGG CGG CAT GAA AGC TGA GAT GGA GGG CGG CAT GAA AGC TGA GAT GGA GGG CGG CAT (Fragment 341) GGC GGG CAC AGG (Fragment 342) GGC GGG CAC AGG (Fragment 343) GGC GGG CAC AGG 23 (Fragment 344) (SEQ. ID NO:351) CAA ACC TGA GAT GGA GGG CCG CAT GGC CCC CAC AGG CTG -3' (Fragment 345) (SEQ. ID NO:352) GAA AGC TCA GAT CCA GGG CGC CAT GGC CCC CAC AGG CT-3' (Fragment 346) (SEQ. ID NO:353) CAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG C-31 (Fragment 347) (SEQ. ID NO:354) GAA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG -3' (Fragment 348) (SEQ. ID NO:355) GAA AGC TGA GAT GGA GCC CGG CAT GGC GGG CAC AG-3' (Fragment 349) (SEQ. ID NO:356) GAA AGC TGA GAT GGA G CGG CAT GCC GGC CAC A-3V (Fragment 350) (SEQ. ID NO:357) GAA ACC TCA GAT CCA GCG CGG CAT GCC GGG CAC-3' (Fragment 351) (SEQ. ID NO:358) 51- CAA AGC TGA CAT CGA GCG CGC CAT CCC CGG CA-3' (Fragment 352) (SEQ. ID NO:359) GAA ACC TGA GAT GGA CCC CCG CAT GGC GGG C-3' (Fragment 353) (SEQ. ID NO:360) GAA ACC TCA CAT GGA GGG CGG CAT GGC CGG -3' (Fragment 354) (SEQ. ID NO:361) GAA AGC TGA CAT GGA GC CGC CAT GGC GG-31 (Fragment 355) (SEQ. ID NO:362) GAA AGC TCA CAT CCA GGG CCC CAT GGC G-31 (Fragment 356) (SEQ. ID NO:363) CAA AGC TCA CAT CCA CCC CGC CAT GGC -3' (Fragment 357) (SEQ. ID NO:364).

51- CAA ACC TGA CAT CCA CCC CCC CAT GG -3' (Fragment 358) (SEQ. ID NO:365) CAA ACC TCA CAT CCA CCC CCC CAT C -3, (Fragment 359) (SEQ. ID NO:366) CAA ACC TGA CAT CGA CCC CGC CAT -3' (Fragment 360) (SEQ. ID NO:367) GAA ACC TCA CAT CCA CCC CGC CA-31 (Fragment 361) (SEQ. ID NO:368) CAA ACC TCA CAT CCA CCC CCC C-3' (Fragment 362) (SEQ. ID NO:369) 24 CAA AGC TGA CAT CCA GCC CGG -3' 5' 5'

GAA

CAA

GAA

CAA

GAA

CAA

TGA

TCA

GGA GCG CG -3' CCA GGC C -3' GGA GCG -3' GCACC -3' CGA C -3' CGA* -3' CC -3' C -3' -3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 363) 364) 365) 366) 367) 368) 369) 370) 371) 372) (SEQ. ID NO:370) (SEQ. ID NO:371) (SEQ. ID NO:372) (SEQ. ID NO:373) (SEQ. ID NO:374) (SEQ. ID NO:375) (SEQ. ID NO:376) (SEQ. ID NO:377) (SEQ. ID NO:378) (SEQ. ID NO:379) (SEQ. ID NO:380) (SEQ. ID NO:381) CTC CCC-3' (SEQ. ID NO:382) CTC CC-3' (SEQ. ID NO:383) CTC C-3' (SEQ. ID NO:384) CTC -3' TCA CA-3' CAA ACC TCA C-3' AA ACC TCA CAT CCA CCC CCC CAT 15 AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT 25 AA ACC TGA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT AA ACC TCA CAT CCA CCC CCC CAT (Fragment 373) (Fragment 374) GGC CCC CAC ACC (Fragment 375) GGC CCC CAC ACC (Fragment 376) GGC CCC CAC ACC (Fragment 377) GQ3C CCC CAC ACC (Fragment 378) CGC CCC CAC ACC C (Fragment 379).

GGC CCC CAC ACC C (Fragment 380) CGC CCC CAC ACC (Fragment 381) GGC CCC CAC AC-3' (Fragment 382) GGC CCC CAC A-3' (Fragment 383) GGC CCC CAC-3' (Fragment 384) GCC CCC CA-3' (Fragment 385) GCC GC C-3' (Fragment 386) (SEQ. ID NO:385) T-3' (SEQ. ID NO:386) -3' (SEQ. ID NO:387) 3' (SEQ. ID NO:388) (SEQ. ID NO:389) (SEQ. ID NO:390) (SEQ. ID NO:391) (SEQ. ID NO:392) (SEQ. ID NO:393) AA AGC TGA GAT GGA 5'- 5'- 5'- 20 5'- 5'-

AGC

TGA

GAT

GGG CGG CAT GGC GGG -3' (Fragment 387) GGG CGG CAT GGC GG-3' (Fragment 388) GGG CGG CAT GGC G-3' (Fragment 389) GGG CGG CAT GGC -3' (Fragment 390) GGG CGG CAT GG -3' (Fragment 391) GGG CGG CAT G -3' (Fragment 392) GGG CGG CAT -3' (Fragment 393) GGG CGG CA-3' (Fragment 394) GGG CGG C-3' (Fragment 395) GGG CGG (Fragment 396) GGG CG (Fragment 397) GGG C (Fragment 398) GGG (Fragment 399) GG (Fragment 400) G (Fragment 401) (Fragment 402) (Fragment 403) 3' (Fragment 404) (Fragment 405) (Fragment 406)

(SEQ.

N0:395) NO:396) N0:397) NO:398) N0:399) NO:400) NO:401) NO:402) NO:403) NO:404) NO:405) NO:406) NO:407) NO:408) NO:409) NO:410) NO:411) NO:412) (SEQ. ID N0:394) AA AGC TGA GA-3' (SEQ. ID NO:413) 5'- A AGC A AGC A AGC A AGC A AGC

TGA

GAT

GGA

GGG

CGG

CAT

GGC GGG CAC AGG CTG GGC-3' (Fragment 407) (SEQ. ID GGC GGG CAC AGG CTG GG-3' (Fragment 408) (SEQ. ID GGC GGG CAC AGG CTG G-3' (Fragment 409) (SEQ. ID GGC GGG CAC AGG CTG -3' (Fragment 410) (SEQ. ID GGC GGG CAC AGG CT-3' NO:414) NO:415) NO:416) NO: 417) 26 (Fragment 411) (SEQ. ID N0:418) A AGC TGA GAT GGA GGG r r 5'- 5'- 5'- 5'- 5'- AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA AGC TGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA GAT GGA

GGG

CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CAT CGG CA- CGG C-3 CGG -3' CG -3' C -3' -3'

CG

C

GGG CAC AGG C-3' (Fragment 412) (SEQ.

GGG CAC AGG -3' (Fragment 413) GGC GGG CAC AG-3' (Fragment 414) GGC GGG CAC A-3' (Fragment 415) GGC GGG CAC-3' (Fragment 416) GGC GGG CA-3' (Fragment 417) GGC GGG C-3' (Fragment 418) GGC GGG -3' (Fragment 419) GGC GG-3' (Fragment 420) GGC G-3' (Fragment 421) GGC -3' (Fragment 422) GG -3' (Fragment 423) G -3' (Fragment 424) -3' (Fragment 425) 3' (Fragment 426) S (Fragment 427) (Fragment 428) (Fragment 429) (Fragment 430) (Fragment 431) (SEQ. ID NO:420)

(SEQ.

NO:421) N0:422) N0:423) N0:424) N0:425) N0:426), NO:427) N0:428) N0:429) NO:430) NO:431) NO: 432) NO:433) N0:434) N0:435) NO:436) NO:437) NO: 438) NO:419) A AGC TGA GAT GGA GG -3 A AGC TGA GAT GGA G -3' (Fragment 432) (SEQ. ID NO:439) (Fragment 433) (SEQ. ID NO:440) 27 A AGC TGA GAT GGA -3' A AGC TGA GAT GG -3' A AGC TGA GAT G -3' A AGC TGA GAT -3' (Fragment 434) (SEQ. ID NO:441) (Fragment 435) (SEQ. ID NO:442) AGC TGA GAT GGA GGG 5'- 15 5'- 5'- 5'-

AGC

TGA

GAT

GGA

GGG

CGG

CAT

GGC

(Fragment 436) (SEQ. ID (Fragment 437) (SEQ. ID GGG CAC AGG CTG GGC-3' (Fragment 438) (SEQ. ID GGG CAC AGG CTG GG-3' (Fragment 439) (SEQ. ID GGG CAC AGG CTG G-3' (Fragment 440) (SEQ. ID GGG CAC AGG CTG -3' (Fragment 441) (SEQ. ID GGG CAC AGG CT-3' (Fragment 442) (SEQ. ID GGG CAC AGG C-3' NO:443) NO:444) NO: 445) NO:446) N0:447) N0:448) NO:449) (Fragment 443) GGG CAC AGG -3' (Fragment 444) GGG CAC AG-3' (Fragment 445) GGG CAC A-3' (Fragment 446) GGG CAC-3' (Fragment 447) GGG CA-3' (Fragment 448) GGG C-3' (Fragment 449) GGG -3' (Fragment 450) GG-3' (Fragment 451) G-3' (Fragment 452) -3' (SEQ. ID NO:450)

(SEQ.

NO:451) NO:452) NO:453) NO:454) NO:455) NO:456) NO:457) NO:458) NO:459) (Fragment 453) (SEQ. ID NO:460) AGC TGA GAT GGA GGG CGG CAT GG -3' 28 (Fragment 454) CGG CAT G -3' (SEQ. ID NO:461) AGC TGA GAT GGA GGG (Fragment 455) 5'-

AGC

TGA

GAT

GGG

CGG CAT CGG CA-3' CGG C-3' CGG -3' CG -3' C -3' -3' AGC TGA GAT GGA GG -3' -3' 9 to..: 0 So* Sr 0 *000 15 5'-

AGC

TGA

GAT

GGA G GGA -3' GG -3' G -3' GC TGA GAT GGA GGG 5'- 20 5'- 5'- 5'-

GAT

GGA

GGG

CGG

CAT

GGC

(Fragment 456) (Fragment 457) (Fragment 458) (Fragment 459) (Fragment 460) (Fragment 461) (Fragment 462) (Fragment 463) (Fragment 464) (Fragment 465) (Fragment 466) (Fragment 467) GGG CAC AGG CTG (Fragment 468) GGG CAC AGG CTG (Fragment 469) GGG CAC AGG CTG (Fragment 470) GGG CAC AGG CTG (Fragment 471) GGG CAC AGG CT-3 (Fragment 472) GGG CAC AGG C-3' (Fragment 473) GGG CAC AGG -3' (Fragment 474) GGG CAC AG-3' (Fragment 475) GGG CAC A-3' (Fragment 476) GGG CAC-3' (Fragment 477) GGG CA-3'

(SEQ.

GGC-3'

(SEQ.

GG-3'

(SEQ.

G-3'

(SEQ.

-3' N0:462) N0:463) N0:464) NO:465) NO:466) NO:467) NO:468) N0:469) N0:470) N0:471) N0:472) N0:473) N0:474) N0:475) N0:476) NO:477) (SEQ. ID N0:478)

(SEQ.

N0:479) N0:480) NO:481) NO:482) N0:483).

NO:484) GC TGA GAT GGA GGG CGG CAT 29 GC TGA GAT OGA GGG 6: of** :%to so..

00* .9 0 60,0.6 S900o a.

ease.

5'- 5,- 5,- 5'- 5'- 5'- 5'- 5'-

TGA

GAT

OAT

GAT

OAT

GAT

OAT

GAT

OAT

GAT

GGA

GGG

G

GG

GGG

GG

GGG

GG

GGG

GOG

GO

G

(Fragment 478) COG CAT GGC GOG C-3' (Fragment 479) CGG CAT GGC GGG -3' (Fragment 480) COG CAT GGC GG-3' I (Fragment 481) CGO CAT GGC G-3' (Fragment 482) COO CAT 0CC -3' (Fragment 483) CGG CAT OG -3' (Fragment 484) COG CAT 0 -3' (Fragment 485) CGG CAT (Fragment 486) COG CA-3' (Fragment 487) COG C-3' (Fragment 488) COG (Fragment 489) CG (Fragment 490) C (Fragment 491) (Fragment 492) (Fragment 493) (Fragment 494) (Fragment 495)

(SEQ.

(SEQ

ID NO:485) ID NO:496) ID NO:487) ID NO:488) ID NO:489) *ID NO:490) *ID NO: 491) ID NO:492) ID NO:493) ID NO:494) ID NO:495) ID NO:496) ID NO:497) ID NO:498) ID NO:499) ID NO:500) ID NO: 501) ID NO:502) GC TGA OAT OGA -3' GC TOA OAT 00 -3' (Fragment 496) (SEQ. ID NO:503) 5'-

TOA

OAT

GAT

OOA

GA

OOA

OGA

GGA

000 000 000 000 000 000

CG

COO

CGO

COO

GOC

GC

GOC

GC

000 CAC AGO (Fragment 000 CAC AGO (Fragment 000 CAC AGO (Fragment 000 CAC AGO (Fragment 000 CAC AGO (Fragment 000 CAC AGO CTO OOC-3' 497) (SEQ.

CTG 00-3' 498) (SEQ.

CTO 0-3' 499) (SEQ.

CTO -3' 500) (SEQ.

CT-3' 501) (SEQ.

c-3' NO: 504) NO: 505) NO: 506) NO: 507) NO: 508) 30 (Fragment 502) (SEQ. ID NO:509) C TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG -3' (Fragment 503) (SEQ. ID NO:510) C TGA GAT GGA GGG CGG CAT GGC GGG CAC AG-3' (Fragment 504) (SEQ. ID NO:511) C TGA GAT GGA GGG CGG CAT GGC GGG CAC A-3' (Fragment 505) (SEQ. ID NO:512) C TGA GAT GGA GGG CGG CAT GGC GGG CAC-3' (Fragment 506) (SEQ. ID NO:513) C TGA GAT GGA GGG CGG CAT GGC GGG CA-3' (Fragment 507) (SEQ. ID NO:514) C TGA GAT GGA GGG CGG CAT GGC GGG C-3' (Fragment 508) (SEQ. ID NO:515) C TGA GAT GGA GGG CGG CAT GGC GGG -3' 15 (Fragment 509) (SEQ. ID NO:516) C TGA GAT GGA GGG CGG CAT GGC GG-3' (Fragment 510) (SEQ. ID NO:517) C TGA GAT GGA GGG CGG CAT GGC G-3' (Fragment 511) (SEQ. ID NO:518) 20 C TGA GAT GGA GGG CGG CAT GGC -3' (Fragment 512) (SEQ. ID NO:519) C TGA GAT GGA GGG CGG CAT GG -3' S:(Fragment 513) (SEQ. ID NO:520) C TGA GAT GGA GGG CGG CAT G-3' (Fragment 514) (SEQ. ID NO:521) C TGA GAT GGA GGG CGG CAT (Fragment 515) (SEQ. ID NO:522) C TGA GAT GGA GGG CGG CA-3' (Fragment 516) (SEQ. ID NO:523) C TGA GAT GGA GGG CGG C-3' (Fragment 517) (SEQ. ID NO:524) C TGA GAT GGA GGG CGG (Fragment 518) (SEQ. ID N0:525) C TGA GAT GGA GGG CG (Fragment 519) (SEQ. ID NO:526) C TGA GAT GGA GGG C (Fragment 520) (SEQ. ID NO:527) C TGA GAT GGA GGG (Fragment 521) (SEQ. ID NO:528) C TGA GAT GGA GG (Fragment 522) (SEQ. ID NO:529) C TGA GAT GGA G (Fragment 523) (SEQ. ID NO:530) C TGA GAT GGA (Fragment 524) (SEQ. ID NO:531) TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 525) (SEQ. ID NO:532) TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GG-3' 31 (Fragment 526) (SEQ. ID NO:533) 5'- 5'- 15 5'- 20 5'- 25 5'- 5'-

TGA

GAT

GGA

GGG

CGG

CAT GGC GGG CAC AGG CTG G-3' (Fragment 527) (SEQ.

CAT GGC GGG CAC AGG CTG -3' (Fragment 528) (SEQ.

CAT GGC GGG CAC AGG CT-3' (Fragment 529) (SEQ.

CAT GGC GGG CAC AGG C-3' (Fragment 530) (SEQ.

CAT GGC GGG CAC AGG -3' (Fragment 531) (SEQ.

CAT GGC GGG CAC AG-3' (Fragment 532) (SEQ.

CAT GGC GGG CAC A-3' (Fragment 533) (SEQ.

CAT GGC GGG CAC-3' (Fragment 534) (SEQ.

CAT GGC GGG CA-3' (Fragment 535) (SEQ.

CAT GGC GGG C-3' (Fragment 536) (SEQ.

CAT GGC GGG -3' (Fragment 537) (SEQ.

CAT GGC GG-3' (Fragment 538) (SEQ.

CAT GGC G-3' (Fragment 539) (SEQ.

CAT GGC -3' (Fragment 540) (SEQ.

CAT GG (Fragment 541) (SEQ.

CAT G (Fragment 542) (SEQ.

CAT (Fragment 543) (SEQ.

CA-3' (Fragment 544) (SEQ.

C-3' (Fragment 545) (SEQ.

(Fragment 546) (SEQ.

NO:534) ID NO:535) NO:536) NO:537) NO:538) NO:539) NO:540) NO:541) NO:542) NO:543) NO:544) NO:545) NO:546) NO:547) NO:548) NO: 549) NO:550) NO:551) NO:552) NO:553) TGA GAT GGA GGG CG -3' TGA GAT GGA GGG C -3' (Fragment 547) (SEQ. ID NO:554) (Fragment 548) (SEQ. ID NO:555) 32- TGA GAT GGA GGG -3' (Fragment 549) (SEQ. ID NO:556) (Fragment 550) (SEQ. ID NO:557) TGA GAT GGA GG TGA GAT GGA G GA GAT GGA GGG 5'- 5'- 20 5'- 5'- 5'-

GAT

GGA

GGG

CGG

CAT

GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GGG GGC GG-: GGC G-3 GGC -3' GG -3' G -3' -3' (Fragment 551) (SEQ.

CAC AGG CTG GGC-3' (Fragment 552) (SEQ.

CAC AGG CTG GG-3' (Fragment 553) (SEQ.

CAC AGG CTG G-3' (Fragment 554) CAC AGG CTG -3' (Fragment 555) CAC AGG CT-3' (Fragment 556) CAC AGG C-3' (Fragment 557) CAC AGG -3' (Fragment 558) CAC AG-3' (Fragment 559) CAC A-3' (Fragment 560) CAC-3' (Fragment 561) CA-3' (Fragment 562) C-3' (Fragment 563) -3' (Fragment 564) 3' (Fragment 565) (Fragment 566) (Fragment 566) (Fragment 567) (Fragment 568) (Fragment 569) (Fragment 570) (SEQ. ID N0:561)

(SEQ.

NO:562) N0:563) NO:564) NO:565) NO:566) N0:567) N0:568) NO:569) NO:570) NO:571) N0:572) NO:573) NO:574) NO:575) NO:576) NO: 577) NO:558) NO:559) NO:560) 5'- 5'- 5'- 15 20 5'- 5'- 5'- GA GAT GGA GGG GA GAT GGA GGG GA GAT GGA GGG 33 CGG CA-3' CGG C-3' CGG -3' GA GAT GGA GGG CG -3' GA GAT GGA GGG GA GAT GGA GGG GA GAT GGA GG A GAT GGA GGG C -3' -3' -3' (Fragment 571) (SEQ.

(Fragment 572) (SEQ.

(Fragment 573) (SEQ.

(Fragment 574) (SEQ.

(Fragment 575) (SEQ.

(Fragment 576) (SEQ.

(Fragment 577) (SEQ.

GAT

GGA

GGG

CGG

CAT

GGC

GGG CAC AGG CTG GGC- (Fragment 578) GGG CAC AGG CTG GG-3 (Fragment 579) GGG CAC AGG CTG G-3' (Fragment 580) GGG CAC AGG CTG -3' (Fragment 581) GGG CAC AGG CT-3' (Fragment 582) GGG CAC AGG C-3' (Fragment 583) GGG CAC AGG -3' (Fragment 584) GGG CAC AG-3' (Fragment 585) GGG CAC A-3' (Fragment 586) GGG CAC-3' (Fragment 587) GGG CA-3' (Fragment 588) GGG C-3' (Fragment 589) GGG -3' (Fragment 590) GG-3' (Fragment 591) G-3' (Fragment 592) 3'

(SEQ.

I

(SEQ. ID N0:586)

(SEQ.

N0:587) N0:588) NO:589) NO:590) N0:591) NO:592) NO:593) NO:594) NO:595) N0:596) NO:597) NO:598) N0:578) N0:579) NO:580) N0:581) NO:582) NO:583) NO:584) NO:585) A GAT GGA GGG CGG CAT GGC (SEQ. ID NO:599) A GAT GGA GGG CGG CAT GGC -3' (Fragment 593) (SEQ. ID NO:600) 34- A GAT GGA GGG CGG CAT GG -3' A GAT GGA GGG CGG CAT G -3' (Fragment 594) (Fragment 595) (SEQ. ID NO:601) (SEQ. ID NO:602) 5'-

GGA

CGG CAT -3' CGG CA-3' CGG C-3' CGG -3' (Fragment (Fragment (Fragment (Fragment 596) 597) 598) 599) A GAT GGA GGG CG -3' A GAT GGA GGG C -3' A GAT GGA GGG -3' GAT GGA GGG CGG CAT (Fragment 600) 5'- 20 5'- 5'- 5'- NO:620)

GAT

GGA

GGG

CGG

CAT

GGC

GGG

(Fragment 601) (Fragment 602) CAC AGG CTG GGC-3 (Fragment 603) CAC AGG CTG GG-3' (Fragment 604) CAC AGG CTG G-3' (Fragment 605) CAC AGG CTG -3' (Fragment 606) CAC AGG CT-3' (Fragment 607) CAC AGG C-3' (Fragment 608) CAC AGG -3' (Fragment 609) CAC AG-3' (Fragment 610) CAC A-3' (Fragment 611) CAC-3' (Fragment 612)

(SEQ.

(SEQ. (SEQ.

(SEQ.

(SEQ. (SEQ.

(SEQ.

NO: 603) NO: 604) NO:605) NO: 606) NO:607) NO: 608) NO:609) NO:610) NO:611) NO: 612) NO:613) NO:614) NO:615) NO:616) NO: 617) NO:618) (SEQ. ID NO:619) CA-3' (Fragment 613) (SEQ. ID C-3' (Fragment 614) (SEQ. ID NO:621) -3' (Fragment 615) (SEQ. ID N0:622) GAT GGA GGG CGG CAT GGC GG-3' (Fragment 616) (SEQ. ID NO:623) GAT GGA GGG CGG CAT GGC G-3' (Fragment 617) (SEQ. ID NO:624) 35 CGG CAT GGC -3' GAT GGA GGG (Fragment 618) (SEQ. ID NO:625) (Fragment 619) (SEQ. ID NO:626) a 5'- 5'- 5'- 5'- 5'-

GAT

GGA

CGG CAT G -3' CGG CAT -3' CGG CA-3' CGG C-3' CGG -3' GAT GGA GGG CG -3' GAT GGA GGG C -3' AT GGA GGG CGG CAT

GGA

CAT

GGC

GGG

(Fragment 620) (Fragment 621) (Fragment 622) (Fragment 623) (Fragment 624) (Fragment 625) (Fragment 626) CAC AGG CTG GGC-3 (Fragment 627) CAC AGG CTG GG-3' (Fragment 628) CAC AGG CTG G-3' (Fragment 629) CAC AGG CTG -3' (Fragment 630) CAC AGG CT-3' (Fragment 631) CAC AGG C-3' (Fragment 632) CAC AGG -3' (Fragment 633) CAC AG-3' (Fragment 634) CAC A-3' (Fragment 635) CAC-3' (Fragment 636) CA-3' (Fragment 637) C-3' (Fragment 638) -3' (Fragment 639)

(SEQ.

NO:627) N0:628) NO:629) NO:630) NO:631) NO:632) NO:633) NO:634) N0:635) NO:636) NO:637) NO:638) NO:639) NO:640) NO:641) NO: 642) NO:643) NO:644) NO:645) NO:646) GAT GGA GGG CGG CAT GG -3' AT GGA GGG CGG CAT GGC GG-3' AT GGA GGG CGG CAT GGC G-3' (Fragment 640) (SEQ. ID NO:647) (Fragment 641) (SEQ. ID NO:648) we

S

*5

S

S.

S

S. S S. 55

S

S. *S S

S

5' 15 5' 5- 20 5,- 5'- 25 5'- 5'- AT GGA GGG CGG AT GGA GGG CGG AT GGA GGG CGG AT GGA GGG CGG AT GGA GGG CGG CA-3' AT GGA GGG CGG AT GGA GGG CGG C-3' -3, AT GGA GGG CG -3' T GGA GGG CGG

GGA

GGG

CGG

CAT

GGC

CAT GGC CAT GG CAT G CAT -3' .36 (Fragment 642) (Fragment 643) 3' (Fragment 644) (Fragment 645) (Fragment 646) (Fragment 647) (Fragment 648) (Fragment 649) GGG CAC AGG CTG GGC-3' (Fragment 650) GGG CAC AGG CTG GG-31 (Fragment 651) GGG CAC AGG CTG G-3' (Fragment 652) GGG CAC AGG CTG -3' (Fragment 653) GGG CAC AGG CT-3' (Fragment 654) GGG CAC AGG C-3' (Fragment 655) GGG CAC AGG -3' (Fragment 656) GGG CAC AG-3' (Fragment 657) GGG CAC A-3' (Fragment 658) GGG CAC-3' (Fragment 659) GGG CA-3' (Fragment 660) GGG C-3' (Fragment 661) GGG (Fragment 662) GG-3' (Fragment 663) G-3' (Fragment 664)

(SEQ.

NO: 649) NO: 650) NO: 651) NO: 652) NO: 6 53) NO: 654) NO: 655) NO: 656) NO: 657) NO: 658) NO 659) -NO: 6 NO: 661) NO: 662) NO: 663) NO: 664) NO: 665) NO: 666) NO: 667) NO: 668) NO: 669) NO: 670) NO: 671) GGA GGG CGG CAT GGC 73' T GGA GGG CGG CAT GG -3' (Fragment 665) (SEQ. ID NO:672) f (Fragment 666) (SEQ. ID NO:673) 37 5,- 5'- GGA GGG GGA GGG GGA GGG GGA GGG GGA GGG CGG CAT G -3' CGG CAT -3' CGG CA-3' CGG C-3' CGG -3' 9* 9~.e

'C

9. 9 9 C a.

a GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC GGA GGG CGG CAT GGC GGG CAC (Fragment 667) (Fragment 668) (Fragment 669) (Fragment 670) (Fragment 671) AGG CTG GGC-3' (Fragment 672) AGG CTG GG-3' (Fragment 673) AGG CTG G-3' (Fragment 674) AGG CTG -3' (Fragment 675) AGG CT-3' (Fragment 676) AGG C-31 (Fragment 677) AGG -3' (Fragment 678) AG-3' (Fragment .679) A-3, (Fragment 680)

(SEQ.

(SEQ. ID NO:679) (SEQ. ID NO:680) (SEQ. ID NO:681) (SEQ. ID NO:682) (SEQ. ID NO:683) (SEQ. ID NO:684) (SEQ. ID NO:685) (SEQ. ID NO:686) (SEQ. ID NO:687) (SEQ. ID NO:688) NO: 674) NO: 675) NO: 676) NO: 677) NO: 678) 5'- Poo* :0-

P.

GGA GGG CGG CAT GGC GGG CAC -3' (Fragment 681) GGA GGG CGG CAT GGC GGG CA- 3' 5'- 51- 5'-

GGA

CGG

CAT GGC GGG C-3' CAT GGC GGG -3' CAT GGC GG-3' CAT GGC G-3' CAT GGC -3' CAT GG -3' CAT G -3' CAT -3' CA-3' C-3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 682) 683) 684) 685) 686) 687) 688) 689) 690) 691) 692)

(SEQ.

NO: 689) NO: 6 NO: 691) NO: 692) NO: 693) NO: 694) NO' 6 NO: 696) NO: 697) NO: 698) NO: 699) 38

S

5'- 15 5'- 20 5'- 5'- 5'-

GGG

CGG

CAT

GGC GGG CAC GGC GGG CAC GGC GGG CAC GGC GGG CAC GGC GGG CAC GGC GGG CAC GGC GGG CAC GGC GGG CAC GGC GGG CAC GGC GGG CAC- GGC GGG CA-3 GGC GGG C-3' GGC GGG -3' GGC GG-3' GGC G-3' GGC -3' GG -3' G -3' -3' AGG CTG GGC-3' (Fragment 693) AGG CTG GG-3' (Fragment 694) AGG CTG G-3' (Fragment 695) AGG CTG -3' (Fragment 696) AGG CT-3' (Fragment 697) AGG C-3' (Fragment 698) AGG -3' (Fragment 699) AG-3' (Fragment 700) A-3' (Fragment 701) 3' (Fragment 702) (Fragment 703) (Fragment 704) (Fragment 705) (Fragment 706) (Fragment 707) (Fragment 708) (Fragment 709) (Fragment 710) (Fragment 711)

(SEQ.

NO:700) NO:701) NO:702) NO:703) NO:704) NO:705) NO:706) NO:707) NO:708) NO:709) NO:710) NO:711) NO:712) NO:713) NO:714) NO:715) NO:716) NO:717) NO:718) NO:719) NO:720) NO:721) N0:722) GA GGG CGG CA-3' A GGG CGG CAT GGC GGG CAC AGG A GGG CGG CAT GGC GGG CAC AGG A GGG CGG CAT GGC GGG CAC AGG A GGG CGG CAT GGC GGG CAC AGG (Fragment 712) CTG GGC-3' (Fragment 713) CTG GG-3' (Fragment 714) CTG G-3' (Fragment 715) CTG -3' 39 (Fragment 716) (SEQ. ID NO:723) A GGG CGG CAT GGC GGG CAC AGG CT-3' (Fragment 717) (SEQ. ID NO:724) A GGG CGG CAT GGC GGG CAC AGG C-3' (Fragment 718) (SEQ. ID NO:725) A GGG CGG CAT GGC GGG CAC AGG -3' (Fragment 719) (SEQ. ID NO:726) A GGG CGG CAT GGC GGG CAC AG-31 (Fragment 720) (SEQ. ID NO:727) A GGG CGG CAT GGC GGG CAC A-3' (Fragment 721) (SEQ. ID NO:728) A GGG CGG CAT GGC GGG CAC-31 (Fragment 722) (SEQ. ID NO:729) A GGG CGG CAT GGC GGG CA-3' (Fragment 723) (SEQ. ID NO:730) A GGG CGG CAT GGC GGG C-3' (Fragment 724) (SEQ. ID NO:731) A GGG CGG CAT GGC GGG (Fragment 725) (SEQ. ID NO:732) A GGG CGG CAT GGC GG-31 (Fragment 726) (SEQ. ID NO:733) *51- A GGG CGG CAT GGC G-31 (Fragment 727) (SEQ. ID NO:734) 51- A GGG CGG CAT GGC (Fragment 728) (SEQ. ID NO:735) A GGG CGG CAT GG (Fragment 729) (SEQ. ID NO:736) A GGG CGG CAT G (Fragment 730) (SEQ. ID NO:737) A GGG CGG CAT (Fra gment 731) (SEQ. ID NO:738) GGG CGG CAT GGC GGG CAC AGG CTG GGC-31 .(Fragment 732) (SEQ. ID NO:739) GGG CGG CAT GGC GGG CAC AGG CTG GG-3' (Fragment 733) (SEQ. ID NO:740) GGG CGG CAT GGC GGG CAC AGG CTG G-3' (Fragment 734) (SEQ. ID NO:741) 51- GGG CGG CAT GGC GGG CAC AGG CTG -3' (Fragment 735) (SEQ. ID NO:742) GGG CGG CAT GGC GGG CAC AGG CT-3' (Fragment 736) (SEQ. ID NO:743) GGG CGG CAT GGC GGG CAC AGG C-3' (Fragment 737) (SEQ. ID NO:744) GGG CGG CAT GGC GGG CAC AGG -3' (Fragment 738) (SEQ. ID NO:745) GGG CGG CAT GGC GGG CAC AG-31 (Fragment 739) (SEQ. ID NO:746) 40 5 5'- 5'- GGG CGG GGG CGG GGG CGG GGG CGG GGG CGG GGG CGG GGG CGG GGG CGG GGG CGG GGG CGG

GGC

GGG CAC A-3' GGG CAC-3' GGG CA-3' GGG C-3' GGG -3' GG-3' -3 (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 740) 741) 742) 743) 744) 745) 746) 747)

(SEQ.

(SEQ.-

(SEQ.

NO: 747) NO: 748) NO: 749) NO: 750) NO: 751) NO: 752) NO: 753) NO: 754) NO: 755) NO 756) CAT GG -3' CAT G -3' GG CGG CAT GGC GGG CAC AGG GG CGG CAT GGC GGG CAC AGG GG CGG CAT GGC GGG CAC AGG GG CGG CAT GGC GGG CAC AGG GG CGG CAT GGC GGG CAC AGG GG CGG CAT GGC GGG CAC AGG (Fragment 748) (Fragment 749) CTG GGC-3' (Fragment 750) CTG GG-3' (Fragment 751) CTG G-3' (Fragment 752) C!TG -3' (Fragment 753) CT-3' (Fragment 754) C-3' (SEQ. ID NO:757) (SEQ. ID NO:758) (SEQ. ID NO:759) (SEQ. ID NO:760) (SEQ. ID NO:761) (SEQ. ID NO:762) (Fragment 755) GG CGG CAT GGC GGG CAC AGG -3' 5'- 5'- 5'- 5'-

CGG

CGO

CGG

CAT

GGC GGG CAC AG-3' GGC GGG CAC A-3' GGC G CAC-3' GGC GGG CA-3' GGC GGG C-3' GGC GGG -3' GGC GG-3' GGC G-3' GGC -3' GG -3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 756) 757) 758) 759) 760) 761) 762) 763) 764) 765) 766)

(SEQ.

NO: 763) NO: 764) NO: 765) NO: 766) NO: 767) NO: 768) NO: 769) NO: 770) NO :771) NO: 772) NO: 773) CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 767) CGG CAT GGC GGG CAC AGG CTG GG-31 (SEQ. ID NO:774) 1, 11 41 CGG CAT GGC GGG CAC G CGG CAT GGC GGG CAC (Fragment 768) AGG CTG G-3' (Fragment 769) AGG CTG -3' (Fragment 770) G CGG CAT GGC GGG CAC AGG CT-3' (Fragment 771) G CGG CAT GGC GGG CAC AGG C-3' (Fragmtent 772) G. CGG CAT GGC GGG CAC AGG -3' (SEQ. ID NO:775) (SEQ. ID NO:776) (SEQ. ID NO:777) (SEQ. ID NO:778) (SEQ. ID NO:779) 5'- 15 5'- 5'- 5'- 5'- G CGG G CGG G CGG G CGG G CGG G CGG G CGG G CGG G CGG

CAT

GGC

CAC AG-3' CAC A-3' CAC-3' CA-3' C-3' -3' GG-3' G-3' -3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment GGC-3' (Fragment GG-3' (Fragment G-3' (Fragment 773) 774) 775) 776) 777) 778) 779) 780) 781) 782)

(SEQ.

NO: 780) NO :781) NO: 782) NO: 783) NO: 784) NO 785) NO 786) NO 787) NO: 788) NO 789) CGG CAT GGC GGG CAC AGG CTG CGG CAT GGC GGG CAC AGG CTG CGG CAT GGC GGG CAC AGG CTG 783) (SEQ. ID NO:790) 784) (SEQ. ID NO:791) 785) (SEQ. ID NO:792) 5'- CGG CAT GGC GGG CAC AGG CTG -3' (Fragment 786) (SEQ. ID NO:793) CGG CAT GGC GGG CAC AGG CT- 3' (Fragment 787) (SEQ. ID NO:794) CGG CAT GGC GGG CAC AGG C-31 5'-

CGG

CAT

GGG

CAC AGG -3' CAC AG-3' CAC A-3' CAC-3' (Fragment (Fragment (Fragment (Fragment (Fragment 788) 789) 790) 791) 792)

(SEQ.

NO: 7 NO 79 6) NO: 797) NO: 7 98) NO: 799) CGG CAT GGC GGG CA-3' CGG AT GC GG C-3'(Fragment 793) (SEQ. ID NO:800) 1 i 42 5'- CGG CAT CGG CAT CGG CAT CGG CAT GGC GGG C-3' GGC GGG -3' GGC GG-3' GGC G-3' (Fragment (Fragment 794) 795)

(SEQ.

NO:801) NO:802) NO:803) NO:804) GG CAT GGC GGG CAC AGG CTG GG CAT GGC GGG CAC AGG CTG GG CAT GGC GGG CAC AGG CTG (Fragment 796) (Fragment 797) GGC-3' (Fragment 798) GG-3' (Fragment 799) G-3' (Fragment 800) (SEQ. ID NO:805) (SEQ. ID NO:806) (SEQ. ID NO:807) (SEQ. ID NO:808) GG CAT GGC GGG CAC AGG CTG -3' (Fragment 801) GG CAT GGC GGG CAC AGG CT-3' r r e 5'- 20 5'- 5'-

CAT

GGC

GGG CAC AG GGG CAC AG GGG CAC AG GGG CAC A- GGG CAC-3' GGG CA-3' GGG C-3' GGG -3' GG-3' G C-3 G -3' -3' 3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment GGC-3' (Fragment GG-3' 802) 803) 804) 805) 806) 807) 808) 809) 810) 811)

(SEQ.

NO:809) NO:810) NO:811) NO:812) NO:813) NO:814) NO:815) NO:816) NO:817) NO:818) G CAT GGC GGG CAC AGG CTG G CAT GGC GGG CAC AGG CTG 812) (SEQ. ID NO:819) (Fragment 813) (SEQ. ID NO:820) G CAT GGC GGG CAC AGG CTG G-3' (Fragment 814) (SEQ. ID NO:821) G CAT GGC GGG CAC AGG CTG -3' 5'- 5'-

CAT

GGG

AGG CT-3' AGG C-3' AGG -3' AG-3' A-3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 815) 816) 817) 818) 819) 820)

(SEQ.

NO:822) NO:823) NO:824) NO:825) NO:826) NO:827) G CAT GGC GGG CAC-3' (Fragment 821) (SEQ. ID NO:828) 43 G CAT GGC GGG CA-3' (Fragment 822) (SEQ. ID NO:829) G CAT GGC G CAT GGC GGG C-3' GGG -3' (Fragment (Fragment CTG GGC-31 823) 824)

(SEQ.

ID NO:830) ID NO: 831) CAT GGC GGG CAC AGG (Fragment 825) CAT GGC GGG CAC AGG CTG GG-3' (Fragment 826) CAT GGC GGG CAC AGG CTG G-3' (SEQ. ID NO:832) (SEQ. ID NO:833) (SEQ. ID NO:834) (Fragment 827) a.

5'- 51- 15 5'- 5'- 5'- 5'-

CAT

GGC

GGG

AGG

CT-3' C-3' -3' CAC AG-3' CAC A-3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 828) 829) 830) 831) 832) 833) 834) 835) 836)

(SEQ.

CAT GGC GGG CAC AGG CTG -3' ID NO:835) ID NO:836) ID NO:837) ID NO:838) ID NO:839) ID NO:840) ID NO: 841) ID NO:842) ID NO:843) CAC-3' CA-3' CAT GGC GGG C-3' AT GGC GGG CAC AGG CTG GGC-3' (Fragment 837) (SEQ. ID NO:844) AT GGC GGG CAC AGG CTG GG-31 (Fragment 838) (SEQ. ID NO: 845) AT GGC GGG CAC AGG CTG G-3' 5'- 5'-

GG

GGG

GG

000 GG0

AGG

CTG-3' CT-3' C-3' CAC AGO -3' CAC AG-3' CAC A-3' CAC-3' CA-3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 839) 840) 841) 842) 843) 844) 845) 846) 847)

(SEQ.

NO: 846) NO: 847) NO: 848) NO: 849) NO: 850) NO: 851) NO: 852) NO 853) NO: 854) T GGC GGG CAC AGG CTG GGC-3' (Fragment 848) (SEQ. ID NO:855) T GGC GGG CAC AGG CTG GG-3' (Fragment 849) (SEQ. ID NO: 856) 44 5'- 15 5'- 5'- 5'- 51- 5'- 5'- 5'- 5'- SGGC GGG CAC AGG CTG G-3' GGC GGG CAC AGG CTG -3' GGC GGG CAC AGG CT-3' GGC GGG CAC AGG C-3' GGC GGG CAC AGG -3' SGGC GGG CAC AG-3' SGGC GGG CAC A-3' SGGC GGG CAC-3' GGC GGG CAC AGG CTG GGC-3' GGC GGG CAC AGG CTG GG-3' GGC GGG CAC AGG CTG G-3' GGC GGG CAC AGG CTG -3' GGC GGG CAC AGG CT-3' GGC GGG CAC AGG C-3 GGC GGG CAC AGG -3' GGC GGG CAC AG-3' GGC GGG CAC A-3' GC GGG CAC AGG CTG GGC-3' GC GGG CAC AGG CTG GG-3' GC GGG CAC AGG CTG G-3' GC GGG CAC AGG CTG -3' GC GGG CAC AGG CT-3' GC GGG CAC AGG C-3' GC GGG CAC AGG -3' GC GGG CAC AG-3' C GGG CAC AGG CTG GGC-3' C GGG CAC AGG CTG GG-3' C GGG CAC AGG CTG G-3' C GGG CAC AGG CTG -3' C GGG CAC AGG CT-3' C GGG CAC AGG C-3' C GGG CAC AGG -3' GGG CAC AGG CTG GGC-3' GGG CAC AGG CTG GG-3' GGG CAC AGG CTG G-3' GGG CAC AGG CTG -3' (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 850) 851) 852) 853) 854) 855) 856) 857) 858) 859) 860) 861) 862) 863) 864) 865) 866) 867) 868) 869) 870) 871) 872) 873) 874) 875) 876) 877) 878) 879) 880) 881) 882) 883) 884) 885)

(SEQ.

NO:857) NO:858) N0:859) NO:860) N0:861) NO:862) NO:863) NO:864) N0:865) NO:866) N0:867) N0:868) N0:869) NO:870) NO:871) N0:872) NO:873) N0:874) NO:875) NO:876) NO:877) N0:878) N0:879) NO:880) NO:881) NO:882) NO:883) NO:884) NO:885) NO:886) NO:887) NO:888) N0:889) NO:890) NO:891) NO:892) 5'- 5'- 5- 5,- 5'-

-GOG

GGC-3' GGG CAC AGG CT-3' GO CAC AGG C-3' GG CAC AGG CTG GGC-3' GO CAC AGG CTG GG-3' GO CAC AGG CTG 0-3' GG CAG AGG CTG -3' GG CAC AGG CT-3' G CAC AGG CTG GGC-3' CAC AGO CTO GG-3' G GAC AGG CTG G-3' G CAC AGO CTG -3' CAC AGO CTG GG-3' CAC AOG CTO 00-3' GAG AGO CTO G-3' AC AGG CTO GGC-3' AC AGG CTG GG-3' C AGO CTG GGC-3' GOC CTG GAA AGC TGA GAT OGA 000 (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment (Fragment 886) 887) 888) 889) 890) 891) 892) 893) 894) 895) 896) 897) 898) 899) 900) 901) 902) 0* a.

a COO CAT GG 000 (Fragment 903) SEQ. ID NO:893) SEQ. ID NO:894) SEQ. ID NO:895) SEQ. ID NO:896) SEQ. ID NO:897) (SEQ. ID NO:898) (SEQ. ID NO:899) (SEQ. ID NO:900) (SEQ. ID NO:901) (SEQ. ID NO:902) (SEQ. ID NO:903) (SEQ. ID NO:904) (SEQ. ID NO:905) (SEQ. ID NO:906) (SEQ. ID NO:907) (SEQ. ID NO:908) (SEQ. ID NO:909) GAG AGO CTO (SEQ. ID NO:910) AC AGO CTO GG- (SEQ. ID NO:911) kC AGO CG GOC- (SEQ. ID NO:912) AGO CG OOC-3' (SEQ. ID NO:913) kGO, CTO OG-3' (SEQ. ID NO:914) 3G CG OG-3' (SEQ. ID NO:915) CG GOC-3' (SEQ. ID NO:916) CT0 OGC-3' (SEQ. ID NO:917) TG GG-3' (SEQ. ID NO:918) 20 5'-GC GG CG OAA AG TGA GAT OGA 000 COO CAT GOC 000 3' (Fragment 904) GG CTO OAA AOC TGA OAT OGA 000 COO CAT GG 000 C 3' (Fragment 905) 5' -GG CTO OAA AOC TOA OAT OGA 000 COG CAT GOC 000 GAG (Fragment 906) CG OAA AOC TOA OAT OGA 000 COG CAT GOC 000 GAGC (Fragment *907) -C CTO GAA AG TOA OAT OOA 000 COO CAT OG 000 GAG AC (Fragment 908) 5'-CTO OAA AG TGA OAT OGA 000 COG CAT GG 000 GAG AGO (Fragment 909) -TO GAA AGG TGA OAT OGA 000 COO CAT GGC 000 GAG AOO (Fragment 910) 51-G OAA AOC TOA OAT OGA 000 COO CAT GOC 000 GAG AGO C' (Fragment 911) AG TGA OAT OGA 000 COG CAT GG 000 GAG AGO CTO OG-3' (Fragment 912) (SEQ. ID NO:919) 46 -AA AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 913) (SEQ.

5'1-A AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 914) (SEQ.

5'-AGC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 915) (SEQ.

-GC TGA GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3.' (Fragment 916) (SEQ.

TGA GAT GGA GGG CGG CAT GOC GGG CAC AGG CTG GGC-3' ID'NO: 9 ID NO:921) ID NO:922) ID NO:923) (Fragment 917) GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 918) GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 919) 15 5'-A GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 920) 5'-GAT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 921) -AT GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 922) GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-31 (Fragment 923) 5' -GGA GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 924) 5 1-GA GGG CGG CAT GGC GGG CAC AGG CTG GGC-31 (Fragment 925) GGG CGG CAT GGC GGG CAC AGG CTG GGC-3'.

(Fragment 926) -GGG CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 927) 51-GG CGG CAT GGC GGG CAC AGG CTG GGC-31 Fragment 928) -G CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 929) 5'-CGG CAT GGC GGG CAC AGG CTG GGC-3' (Fragment 930) 5'1-GG CAT GGC GGG CAC AGG CTG GGC- 3, (Fragment 931) CAT GGC GGG CAC AGG CTG GGC-31 (Fragment 932) (SEQ. ID NO:924) (SEQ. ID NO:925) (SEQ. ID NO:926) (SEQ. ID NO:927) (SEQ. ID NO:928) (SEQ. ID NO:929) (SEQ. ID NO:930) (SEQ. ID N'O:931) (SEQ. ID NO:932) (SEQ. ID NO:933) (SEQ. ID NO:934) (SEQ. ID NO:935) (SEQ. ID NO:936) (SEQ. ID NO:937) (SEQ. ID NO:938) (SEQ. ID NO:939) 47 GGC GGG CAC AGG CTG GGC-3' GGC GGG CAC AGG CTG GGC-3' GGC GGG CAC AGG CTG GGC-3' GGG CAC AGG CTG GGC-3' 5'-GC GGG CAC AGG CTG GGC-3' GGG CAC AGG CTG GGC-3' CAC AGG CTG GGC-3' CAC AGG CTG GGC-3' CAC AGG CTG GGC-3' 5'-CAC AGG CTG GGC-3' AGG CTG GGC-3' AGG CTG GGC-3' CTG GGC-3' (Fragment 933) (Fragment 934) (Fragment 935) (Fragment 936) (Fragment 937) (Fragment 938) (Fragment 939) (Fragment 940) (Fragment 941) (Fragment 942) (Fragment 943) (Fragment 944) (Fragment 945)

(SEQ.

ID NO:940) ID NO:941) ID NO:942) ID NO:943) ID NO:944) ID NO:945) ID NO:946) ID N0:947) ID NO:948) ID NO:949) ID NO:950) ID NO:951) ID N0:952) too.

to. 0 00.* .000 0 *o o so a 15 In the antisense oligonucleotides of the present invention, exemplified by the preceding sequences, adenosine bases may be replaced with an appropriate "spacer" or universal base deoxyribofuranosyl]-5-nitroindole], or with an adenosine agonist or antagonist that does not stimulate adenosine Ai or A 3 receptors. Also part of this invention are chemical analogs of oligonucleotides in which, for example, the phosphodiester bonds have been modified, to a methylphosphonate, a phosphotriester, a phosphorothioate, 25 a phosphorodithioate, or a phosphoramidate, so as to render the oligonucleotide more stable in vivo. The naturally occurring phosphodiester linkages in oligonucleotides are susceptible to degradation by endogenously occurring cellular nucleases, while many analogous linkages are highly resistant to nuclease degradation. See Milligan et al., and Cohen, J. supra. The use of a 3 -end cap" strategy by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the oligonucleotide protects oligonucleotides from degradation. See Tidd, D. M. and Warenius, Br. J.

Cancer 60, 343-350 (1989); Shaw, J.P. et al., Nucleic Acids Res. 19, 747-750 (1991). Phosphoramidate, phosphorothioate, 48 and methylphosphonate linkages are suitable for use in this invention. In addition, extensive modification of the phosphodiester backbone has been shown to impart stability and may allow for enhanced affinity and increased cellular permeation of oligonucleotides. See Milligan, et al., supra. Many different chemical strategies have been employed to replace the entire phosphodiester backbone with novel linkages. Id. The analogues of the oligonucleotides of the invention include phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, boranophosphate, phosphotriester, formacetal, 3'thioformacetal, 5'-thioformacetal, thioether, carbonate, 5'-N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, 15 hydroxylamine, methylene (methylimino) (MMI) or methyleneoxy (methylimino) (MOMI) linkages.

Phosphorothioate and methylphosphonate-modified oligonucleotides are particularly preferred because of their availability and suitability for automated 20 oligonucleotide synthesis. Id. Antisense oligonucleotides containing modifications to the nucleotide base itself a C-5 propyne) or to the sugar a carbohydrate modification), are also aspects of the present invention.

Where appropriate, the antisense nucleotide may be administered in the form of pharmaceutically acceptable salts.

Antisense oligonucleotides may be of any suitable length, from about 10 to 60 nucleotide in length, depending on the particular target being bound and their mode of delivery. Preferably the antisense oligonucleotide is directed to an mRNA region containing a junction between intron and exon. Where the antisense oligonucleotide is directed to an intron/exon junction, it may either entirely overlie the junction or may be sufficiently close to the junction to inhibit the splicing out of the intervening exon during processing of precursor mRNA to mature mRNA, with the 3' or 5' terminus of the antisense 49 oligonucleotide being positioned within about, for example, 5, 3, or 2 nucleotide of the intron/exon junction. Also preferred are antisense oligonucleotides which overlap the initiation codon.

When practicing the present invention, the antisense oligonucleotides administered may be related in origin to the species to which it is administered. When treating humans, the antisense may be derived from human sequences.

Pharmaceutical compositions provided herein comprise an antisense oligonucleotide as given above.

These compositions are administered in amounts effective to reduce the expression of an adenosine receptor, such as the A or A 3 adenosine receptor by passing through a cell 15 membrane and binding specifically with mRNA encoding an Ai or A 3 adenosine receptor in the cell and prevent its translation. Such compositions are provided in a suitable pharmaceutically acceptable carrier sterile

S

pyrogen-free saline solution. The antisense 20 oligonucleotides may additionally be formulated with a hydrophobic carrier capable of passing through a cell membrane, in a liposome, with the liposomes carried in a pharmaceutically acceptable aqueous carrier. The 0 oligonucleotides may also be coupled to a substance which inactivates mRNA, such as a ribozyme. The present oligonucleotides may be administered to a subject aflicted with any disease or condition associated with the lung adenosine receptors to inhibit the activation of Ai or A 3 adenosine receptors. The pharmaceutical formulation may also contain chimeric molecules comprising antisense oligonucleotides attached to molecules which are known to be internalized by cells. These oligonucleotide conjugates utilize cellular uptake pathways to increase the cellular-...

concentrations of oligonucleotides. Examples of macromolecules used in this manner include transferrin, asialoglycoprotein (bound to oligonucleotides via polylysine) and streptavidin.

50 In the pharmaceutical formulation the antisense compound may be contained within a lipid particle or vesicle, such as a liposome or microcrystal. The lipid particles may be of any suitable structure, such as unilamellar or plurilamellar, so long as the antisense oligonucleotide is contained therein. Positively charged lipids such as N- 3 -dioleoyloxi) propyl] N, Ntrimethyl-ammoniumethylsulfate, or "DOTAP," are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, U.S. Patent Nos. 4,880,635 to Janoff et al.; 4,906,477 to Kurono et al.; 4,911,928 to Wallach; 4,917,951 to Wallach; 4,920,016 to Allen et al.; 4,921,757.

The composition of the invention may be 15 administered by any means which transports the antisense nucleotide composition to the lung. The antisense compounds disclosed herein may be administered to the lungs of a patient by any suitable means, but are preferably administered by inhalation of an aerosol comprised of respirable particles which comprise the antisense compound.

The respirable particles may be liquid or solid, and they may optionally contain other therapeutic ingredients.

The antisense compound of the present invention S* should be administered as a formulation including particles 25 of respirable size: that is, particles of a size sufficiently small to pass through the nose, mouth and larynx upon inhalation and through the bronchi and alveoli of the lungs. In general, respirable particles range from about .5 to 10 microns in size. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of nonrespirable particles in the aerosol is thus minimized. For nasal administration, a particle size in the range of 500 im is preferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing an aerosol may be prepared by 51 combining the antisense compound with a suitable vehicle, such as sterile pyrogen free water. Other therapeutic compounds may optionally be included.

Solid particulate compositions containing respirable dry particles of micronized antisense compound may be prepared by grinding dry antisense compound with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising of the antisense compound may optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which may be blended with the antisense compound in any suitable ratio, 15 a 1 to 1 ratio by weight.

The antisense compound may be administered in amount which depends upon the disease being treated, the condition of the subject, the particular formulation, the route of administration, the timing of administration to a subject, etc. In general, intracellular concentrations of the oligonucleotide of from .05 to 50 iM, or more particularly .2 to 5 IM, are desired. For administration to a subject such as a human, a dosage of about .01, .1, or 1 mg/Kg up to 50, 100, or 150 mg/Kg or more is typically employed. Depending on the solubility of the particular formulation of active compound administered, the daily dose may be divided among one or several unit dose administrations. The administration of the antisense compounds may be carried out therapeutically, as a rescue treatment, or prophylactically.

The aerosols of liquid particles comprising the antisense compound may be produced by any suitable means, such as with a nebulizer. See, U.S. Patent No.

4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or 52 oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants.

The aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate medicament aerosol generator. Aerosol .15 generators for administering solid particulate medicaments to a subject produce particles which are respirable,' as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at .a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by I"insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder, a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation.

A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are 53 pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 150 il, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more cosolvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.

15 The aerosol, whether formed from solid or liquid particles, may be produced by the aerosol generator at a rate of from about 10 to 150 liters per minute, more preferably from about 30 to 150 liters per minute, and most preferably about 60 liters per minute. Aerosols containing greater amounts of medicament may be administered more rapidly.

The following examples are provided to illustrate the present invention, and should not be construed as limiting thereon. In these examples, iM means micromolar, 25 mL means milliliters, im means micrometers, mm means millimeters, cm means centimeters, OC means degrees Celsius, ig means micrograms, mg means milligrams, g means grams, kg means kilograms, M means molar, and h means hours.

54 Example 1: Design and synthesis of antisense oligonucleotides The design of antisense oligonucleotides against the AI and A 3 adenosine receptors may require the solution of the complex secondary structure of the target Al receptor mRNA and the target A 3 receptor mRNA. After generating this structure, antisense nucleotides are designed which target regions of mRNA which might be construed to confer functional activity or stability to the mRNA and which optimally may overlap the initiation codon.

Other target sites are readily usable. As a demonstration of specificity of the antisense effect, other oligonucleotides not totally complementary to the target mRNA, but containing identical nucleotide compositions on a 15 w/w basis, are included as controls in antisense .:...experiments.

Adenosine Ai receptor mRNA secondary structure was analyzed and used as described above to design a phosphorothioate :.antisense oligonucleotide. The antisense oligonucleotide which was synthesized was designated HAdAlAS and had the following sequence: 5' -GAT GGA GGG CGG CAT GGC GGG-3' (SEQ ID NO:1) 25 As a control, a mismatched phosphorothioate antisense nucleotide designated HAdAlMM was synthesized with the following sequence: -GTA GCA GGC GGG GAT GGG GGC-3' (SEQ ID NO:2) Each oligonucleotide had identical base content and general sequence structure. Homology searches in GENBANK (release 85.0) and EMBL (release 40.0) indicated that the antisense oligonucleotide was specific for the human and rabbit adenosine Ai receptor genes, and that the mismatched control was not a candidate for hybridization with any known gene sequence.

1 55 Adenosine A 3 receptor mRNA secondary structure was similarly analyzed and used as described above to design two phosphorothioate antisense oligonucleotides.

The first antisense oligonucleotide (HAdA3ASl) synthesized had the following sequence: -GTT GTT GGG CAT CTT GCC-3' (SEQ ID NO:3) As a control, a mismatched phosphorothioate antisense oligonucleotide (HAdA3MM1) was synthesized, having the following sequence: 5' -GTA CTT GCG GAT CTA GGC-3' (SEQ ID NO:4) 15 A second phosphorothioate antisense oligonucleotide (HAdA3AS2) was also designed and synthesized, having the following sequence: 5' -GTG GGC CTA GCT CTC GCC-3' (SEQ ID Its control oligonucleotide (HAdA3MM2) had the sequence: -GTC GGG GTA CCT GTC GGC-3' (SEQ ID NO:6) 25 Phosphorothioate oligonucleotides were synthesized on an Applied Biosystems Model 396 Oligonucleotide Synthesizer, and purified using NENSORB chromatography (DuPont, MD).

Example 2: Testing of Al-Adenosine Receptor Antisense Oligonucleotides in vitro The antisense oligonucleotide against the human AI receptor (SEQ ID NO:1) described above was tested for efficacy in an in vitro model utilizing lung adenocarcinoma cells HTB-54. HTB-54 lung adenocarcinoma cells were demonstrated to express the Ai adenosine receptor using standard northern blotting procedures and receptor probes designed and synthesized in the laboratory.

56 HTB-54 human lung adenocarcinoma cells (106/100 mm tissue culture dish) were exposed to 5.0 iM HAdAlAS or HAdA1MM for 24 hours, with a fresh change of media and oligonucleotides after 12 hours of incubation. Following 24 hour exposure to the oligonucleotides, cells were harvested and their RNA extracted by standard procedures.

A 21-mer probe corresponding to the region of mRNA targeted by the antisense (and therefore having the same sequence as the antisense, but not phosphorothioated) was synthesized and used to probe northern blots of RNA prepared from HAdAlAS-treated, HAdA1MM-treated and non-treated HTB-54 cells. These blots showed clearly that HAdAlAS but not HAdAMM effectively reduced human adenosine receptor mRNA This result showed that HAdAlAS is a good 15 candidate for an anti-asthma drug since it depletes intracellular mRNA for the adenosine Ai receptor, which is involved in asthma.

Example 3: Efficacy of A, Adenosine Receptor Antisense Oligonucleotides in vivo A fortuitous homology between the rabbit and human DNA sequences within the adenosine Ai gene overlapping the initiation codon permitted the use of the phosphorothioate antisense oligonucleotides initially 25 designed for use against the human adenosine Ai receptor in a rabbit model.

Neonatal New Zealand white Pasteurella-free rabbits were immunized intraperitoneally within 24 hours of birth with 312 antigen units/mL house dustmite farinae) extract (Berkeley Biologicals, Berkeley, CA), mixed with kaolin. Immunizations were repeated weekly for the first month and then biweekly for the next 2 months. At 3- 4 months of age, eight sensitized rabbits were anesthetized and relaxed with a mixture of ketamine hydrochloride (44 mg/kg) and acepromazine maleate (0.4 mg/kg) administered intramuscularly.

The rabbits were then laid supine in a 57 comfortable position on a small molded, padded animal board and intubated with a 4.0-mm intratracheal tube (Mallinkrodt, Inc., Glens Falls, NY). A polyethylene catheter of external diameter 2.4 mm with an attached latex balloon was passed into the esophagus and maintained at the same distance (approximately 16 cm) from the mouth throughout the experiments. The intratracheal tube was attached to a heated Fleisch pneumotachograph (size 00; DOM Medical, Richmond, VA), and flow was measured using a Validyne differential pressure transducer (Model DP- 45161927; Validyne Engineering Corp., Northridge, CA) driven by a Gould carrier amplifier (Model 11-4113; Gould Electronic, Cleveland, OH). The esophageal balloon was attached to one side of the differential pressure 15 transducer, and the outflow of the intratracheal tube was connected to the opposite side of the pressure transducer to allow recording of transpulmonary pressure. Flow was integrated to give a continuous tidal volume, and measurements of total lung resistance (RL) and dynamic compliance (Cdyn) were calculated at isovolumetric and flow zero points, respectively, using an automated respiratory o analyzer (Model 6; Buxco, Sharon, CT).

Animals were randomized and on Day 1 pretreatment values for PC50 were obtained for aerosolized adenosine.

25 Antisense (HAdAlAS) or mismatched control (HAdAMM) oligonucleotides were dissolved in sterile physiological saline at a concentration of 5000 ig (5 mg) per 1.0 ml.

Animals were subsequently administered the aerosolized antisense or mismatch oligonucleotide via the intratracheal tube (approximately 5000 ig in a volume of 1.0 ml), twice daily for two days. Aerosols of either saline, adenosine, or antisense or mismatch oligonucleotides were generated by an ultrasonic nebulizer (DeVilbiss, Somerset, PA), producing aerosol droplets 80% of which were smaller than im in diameter.

In the first arm of the experiment, four randomly selected allergic rabbits were administered antisense 9 9 58 oligonucleotide and four the mismatched control oligonucleotide. On the morning of the third day, values (the concentration of aerosolized adenosine in mg/ml required to reduce the dynamic compliance of the bronchial airway 50% from the baseline value) were obtained and compared to PC50 values obtained for these animals prior to exposure to oligonucleotide.

Following a 1 week interval, animals were crossed over, with those previously administered mismatch control oligonucleotide now administered antisense oligonucleotide, and those previously treated with antisense oligonucleotide now administered mismatch control oligonucleotide.

Treatment methods and measurements were identical to those employed in the first arm of the experiment. It should be noted that in six of the eight animals treated with antisense oligonucleotide, adenosine-mediated bronchoconstriction could not be obtained up to the limit of solubility of adenosine, 20 mg/ml. For the purpose of calculation, PC50 values for these animals were set at mg/ml. The values given therefore represent a minimum figure for antisense effectiveness. Actual effectiveness was higher. The results of this experiment are illustrated in both Figure 1 and Table 1.

Table 1. Effects Of Adenosine Ai Receptor Antisense Oligonucleotide Upon Pc50 Values In Asthmatic Rabbits Mismatch Control Ai Receptor Antisense Oligonucleotide Pre Post Pre Post oligonucleotide oligonucleotide oligonucleotide oligonucleotide 3.56 1.02 5.16 1.93 2.36 0.68 >19.5 0.34** Results are presented as the mean (N 8) SEM. Significance was determined by repeated-measures analysis of variance (ANOVA), and Tukey's protected t test. **Significantly different from all other groups, P 0.01.

59 In both arms of the experiment, animals receiving the antisense oligonucleotide showed an order of magnitude increase in the dose of aerosolized adenosine required to reduce dynamic compliance of the lung by 50%. No effect of the mismatched control oligonucleotide upon PC50 values was observed. No toxicity was observed in any animal receiving either antisense or control inhaled oligonucleotide.

These results show clearly that the lung has exceptional potential as a target for antisense oligonucleotide-based therapeutic intervention in lung disease. They further show, in a model system which closely resembles human asthma, that downregulation of the adenosine A, receptor largely eliminates adenosine-mediated bronchoconstriction in asthmatic airways. Bronchial 15 hyperresponsiveness in the allergic rabbit model of human asthma is an excellent endpoint for antisense intervention since the tissues involved in this response lie near to the point of contact with aerosolized oligonucleotides, and the model closely simulates an important human disease.

S• Example 4: Specificity of A-adenosine receptor Antisenseoligonucleotide At the conclusion of the crossover experiment of S: Example 3, airway smooth muscle from all rabbits was quantitatively analyzed for adenosine A, receptor number.

As a control for the specificity of the antisense oligonucleotide, adenosine A 2 receptors, which should not have been affected, were also quantified.

Airway smooth muscle tissue was dissected from each rabbit and a membrane fraction prepared according to described methods Kleinstein and H. Glossmann, Naunyn- Schmiedeberg's Arch. Pharmacol. 305, 191-200 (1978), with slight modifications. Crude plasma membrane preparations were stored at -70 0 C until the time of assay. Protein content was determined by the method of Bradford (M.

Bradford, Anal. Biochem. 72, 240-254 (1976)). Frozen plasma membranes were thawed at room temperature and were rb 60 incubated with 0.2 U/ml adenosine deaminase for 30 minutes at 37 0 C to remove endogenous adenosine. The binding of 3 H] DPCPX (At receptor-specific) or [3H] CGS-21680 (A 2 receptor-specific) was measured as previously described. S.

Ali et al., J. Pharmacol. Exp. Ther. 268, 1328-1334 (1994); S. Ali et al., Am. J. Physiol. 266, L271-277 (1994).

As illustrated in both Figure 2 and Table 2, animals treated with adenosine Ai antisense oligonucleotide in the crossover experiment had a nearly 75% decrease in Ai receptor number compared to controls, as assayed by specific binding of the Al-specific antagonist DPCPX.

There was no change in adenosine A 2 receptor number, as assayed by specific binding of the A 2 receptor-specific agonist 2- (2-carboxyethyl)-phenethylamino] 15 ethylcarboxamido) adenosine (CGS-21680).

Table 2. Specificity Of Action Of Adenosine Ai Receptor Antisense Oligonucleotide 20 Mismatch Control Al Antisense Oligonucleotide Oligonucleotide Al-Specific Binding 1105 48** 293 18

A

2 -Specific Binding 302 22 442 171 a Results are presented as the mean (N 8) SEM. Significance was o" determined by repeated-measures analysis of variance (ANOVA), and Tukey's protected t test. **Significantly different from mismatch control, P 0.01.

The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

The entire disclosure in the complete specification of our Australian Patent Application No.

60295/96 is by cross reference incorporated into the present specification.

Claims

1. A pharmaceutical composition, comprising an oligonucleotide (oligo), which is effective for reducing levels of or sensitivity to adenosine, or for alleviating bronchoconstriction, lung inflammation and/or allergy, and is antisense to the initiation codon, the coding region or the 5' and 3' intron-exon junctions of a gene encoding the adenosine Ai receptor or antisense to an adenosine Ai receptor mRNA; and a pharmaceutical carrier.

2. A pharmaceutical composition according to claim 1, wherein the oligo is linked or modified by at least one of methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino), methyleneoxy (methylimino) or phosphoramidate residues. 20 3. A pharmaceutical composition according to claim 2, wherein all bases in the oligos are linked or modified S. by methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, o thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino), .oo methyleneoxy (methylimino) and/or phosphoramidate residues.

94. A pharmaceutical composition according to any one of claims 1 to 3, wherein the oligo is a DNA. 5. A pharmaceutical composition according to any one of claims 1 to 3, wherein the oligo is an RNA. 6. A pharmaceutical composition according to any one of claims 1 to 3, wherein the oligo is antisense to the initiation codon of a gene encoding an adenosine Ai receptor or antisense to an adenosine Ai receptor mRNA. 7. A pharmaceutical composition according to any one of claims 1 to 6, wherein the oligo comprises about 10 to H:\Bkrot\Keep\speci\EPI-0796b.doc 26/07/00 62 up to about 60 mononucleotides. 8. A pharmaceutical composition according to claim 7, wherein the oligo comprises about 18 up to about 21 mononucleotides. 9. A pharmaceutical composition according to claim 1, wherein the oligo is antisense to the 3' intron-exon junction of an adenosine Ai receptor gene or antisense to an adenosine Ai receptor mRNA. A pharmaceutical composition according to claim 1, wherein the oligo is antisense to the 5' intron-exon junction of an adenosine Ai receptor gene or antisense to an adenosine Ai receptor mRNA. 11. A pharmaceutical composition according to claim 1, wherein the oligo is antisense to the coding region of a gene encoding the adenosine Ai receptor or antisense to an adenosine Ai receptor mRNA. 12. A pharmaceutical composition according to claim 1, wherein the oligo is SEQ. ID NO: 1, SEQ. ID NO:3, SEQ. ID NO:5 or SEQ. ID NO:7 to SEQ. ID NO:952; or SEQ. ID NO:1, to 20 SEQ. ID,NO:3, SEQ. ID NO:5, or SEQ. ID NO:7 to SEQ. ID S" NO:952, wherein the oligo is linked or modified by at least one of methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, :oo: formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, :methylene(methyimino), methyleneoxy (methylimino) or phosphoramidate residues. 13. A pharmaceutical composition according to claim 12, wherein the oligo is selected from SEQ. ID NO: 7 to SEQ. ID NO:952; or SEQ. ID NO: 7 to SEQ. ID NO:952, wherein the oligo is linked or modified by at least one of methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino), H:\Bkrot\Keep\speci\EPI-0796b.doc 26/07/00 63 methyleneoxy (methylimino) or phosphoramidate residues. 14. A pharmaceutical composition according to claim 12, wherein the oligo is selected from SEQ. ID NO:1, SEQ. ID NO:3, SEQ. ID NO:5 or SEQ. ID NO:7 to SEQ. ID NO:952. 15. A pharmaceutical composition according to claim 14, wherein the oligo is SEQ. ID NO: 7. 16. A pharmaceutical composition according to claim wherein the oligo is SEQ. ID NO: 7 and all mononucleotide linking residues are phosphorothioate residues. 17. A pharmaceutical composition according to claim 12, wherein the oligo is selected from the group consisting of SEQ. ID NO:1, SEQ. ID NO:3, SEQ. ID NO:5, or SEQ. ID NO:7 to SEQ. ID NO:952, wherein the oligo is linked or substituted by at least one of methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, 20 hydroxylamine, methylene(methyimino), methyleneoxy (methylimino) or phosphoramidate residues. 18. A pharmaceutical composition according to claim 14, wherein the oligo is selected from SEQ. ID NO:8 to SEQ. ID NO:952. 19. A pharmaceutical composition according to claim 18, wherein the oligo is selected from SEQ. ID NO:8 to SEQ. ID NO:952 and at least one mononucleotide linking residue is a phosphorothioate residue. A pharmaceutical composition according to claim 30 19, wherein all mononucleotide linking residues are phosphorothioate residues. 21. A pharmaceutical composition according to any one of claims 1 to 20, wherein the carrier is selected from the group consisting of solid and liquid carriers. 22. A pharmaceutical composition according to any one of claims 1 to 21, further comprising an agent selected from the group consisting of antioxidants, flavoring H:\Bkrot\Keep\speci\EPI-0796b.doc 26/07/00 64 agents, a mRNA inactivator, a the molecule capable of cellular uptake, volatile oils, buffering agents, dispersants, surfactants, propellants and preservatives. 23. A pharmaceutical composition according to claim 22, wherein the mRNA inactivator comprises an enzyme. 24. A pharmaceutical composition according to claim 22, wherein the enzyme comprises a ribozyme. A pharmaceutical composition according to claim 22, wherein the molecule capable of cellular uptake is a macromolecule selected from the group consisting of transferrin, asialoglycoprotein, asialoglycoprotein bound to the oligonucleotide via polylysine and streptavidin. 26. A pharmaceutical composition according to any one of claims 1 to 25, wherein the oligo is present in an amount of about 0.1 to about 100 w/w of the composition. 27. A pharmaceutical composition according to claim 26, wherein the oligo is present in an amount of about 0.1 up to about 40% w/w of the composition. 28. A pharmaceutical composition according to claim 20 27, wherein the oligo is present in an amount of about 0.1 up to about 20 w/w of the composition. 29. A pharmaceutical composition according to any one of claims 1 to 28, wherein the carrier comprises a hydrophobic carrier. 30. A pharmaceutical composition according to claim 29, wherein the carrier comprises lipid particles or vesicles. 31. A pharmaceutical composition according to claim wherein the vesicles comprise liposomes and the S 30 particles comprise microcrystals. 32. A pharmaceutical composition according to any one of claims 1 to 31, which is a systemic formulation. 33. A pharmaceutical composition according to any one of claims 1 to 31, which is a nasal formulation or a formulation comprising liquid or solid respirable particles. 34. A pharmaceutical composition according to claim H:\Bkrot\Keep\speci\EPI-O796b.doc 26/07/00 65 33, comprising oligo particles of about 0.5 to about 10 Imn in size. A pharmaceutical composition according to claim 33, comprising oligo particles of about 10 to--about 500 rn in size. 36. A pharmaceutical composition according to any one of claims 33 'to 35, which is an aerosol formulation. 37. A pharmaceutical composition according to any one of claims 33 to 36, comprised in a capsule or cartridge. 38. A pharmaceutical composition according to claim 33, comprising a suspension or solution of liquid or solid particles of the oligo in a solvent or mixture of solvents. 39. A host cell, comprising a oligo according to claim 36. 40. A composite, comprising a delivery device and, in a separate container, a composition according to any one of claims 1 to 38. 41. A composite according to claim 40, wherein the delivery device comprises an inhalator which delivers 20 individual pre-metered doses of the composition. 42. A composite according to claim 40 or claim 41, .i."further comprising an agent selected from the group consisting of solvents, surfactants, antioxidants, surfactant, therapeutic compounds and flavouring agents. 43. A composite according to any one of claims 40 to 42, further comprising a container selected from the group S: consisting of capsules and cartridges, and wherein the composition is provided inside the capsule or cartridge. 44. A composite according to any one of claims 40 to 43, wherein the inhalator comprises a nebulizer which delivers single metered doses of the formulation. 45. A composite according to claim 44, wherein the nebulizer comprises an insufflator; and the composition is provided in a piercable or openable capsule or cartridge. 46. A composite according to claim 44, wherein the delivery device comprises a pressurized inhaler; and the composition comprises a suspension, solution or dry H:\Bkrot\Keep\speci\EPI-0796b.doc 26/07/00 66 formulation of the agent in a solvent. 47. A composite according to claim 46, wherein the solvent is selected from the group consisting of organic solvents and organic solvents mixed with one or more co- solvents. 48. A composite according to claim 41, wherein the inhalator comprises an insufflator; and further comprising a piercable or openable capsule or cartridge with solid particles of the oligo. 49. A composite according to claim 40, wherein the delivery device comprises a pressurized inhaler; and the formulation comprises a suspension or solution of the oligo. A composite according to claim 40, further comprising a propellant and means for delivery thereof; and instructions for preparation and delivery of a composition comprising particles of about 0.5 to about 10 pm and about to about 50 1m in size of the antisense oligo with the propellant means. 51. A method of reducing sensitivity to or levels of adenosine, bronchoconstriction, lung inflammation or allergy or of treating a disease or condition associated with either of them, comprising administering to the respiration of a subject in need of treatment a pharmaceutical composition according to any one of claims 1 o: to 38. 52. A method according to claim 51, wherein the pharmaceutical composition is administered by inhalation, into the lung or nasally. 53. A method according to claim 51 or claim 52, wherein the disease or condition comprises a respiratory disease or condition associated with bronchoconstriction, lung inflammation or allergy. 54. A method according to claim 53, wherein the disease or condition comprises lung inflammation. A method according to any one of claims 51 to 54, wherein the disease or condition comprises asthma. H:\Bkrot\Keep\speci\EpI-079 6 b-doc 26/07/00 67 56. A method according to any one of claims 51 to wherein the subject is non-human. 57. A method according to any one of claims 51 to wherein the subject is a human. 58. A method according to any one of claims 51 to 57, wherein the oligo is administered in an amount of about 0.01 to about 115 mg/kg body weight. 59. A method according to any one of claims 51 to 57, wherein the oligo is administered in an amount of about 1 to about 100 mg/kg body weight. A method according to any one of claims 51 to 57, wherein the oligo is administered in an amount of about up to about 15 mg/kg body weight. 61. A method according to any one of claims 51 to being a prophylactic or therapeutic method. 62. A method according to any one of claims 51 to 61, further comprising in the composition an agent selected from the group consisting of antioxidants, flavouring agents, volatile oils, buffering agents, dispersants, surfactants, propellants and preservatives. 63. A method according to claim 62, wherein the pharmaceutical composition comprises a surfactant. 64. An in vivo method of delivering an oligonucleotide (oligo) to a target polynucleotide associated with increased sensitivity to or levels of adenosine, bronchoconstriction, inflammation or allergy or a disease or condition associated with either of them, comprising administering to a subject a composition according to any one of claims 1 to 38. 30 65. A method according to claim 64, wherein the disease or condition comprises asthma. 66. A method according to claim 64 or claim wherein the subject is a human. 67. A method according to claim 64 or claim wherein the subject is a non-human mammal. 68. A method according to any one of claims 64 to 68, wherein the oligo is administered in an amount of about H:\Bkrot\Keep\Speci\EPIO179 6 b6doc 26/07/00 68 0.01 to about 115 mg/kg body weight. 69. A method according to claim 68, wherein the oligo is administered in an amount of about 1 to about 100 mg/kg body weight. 70. A method according to claim 69, wherein the oligo is administered in an amount of about 1 up to about mg/kg body weight. 71. A method according to any one of claims 64 to being a prophylactic or a therapeutic method. 72. Use of an oligonucleotide (oligo) to reduce sensitivity to or levels of adenosine, bronchoconstriction, lung inflammation, or allergy or of treating a disease or condition associated with either of them, wherein the oligo is antisense to the initiation codon, the coding region or the 5' and 3' intron-exon junctions of a gene encoding the adenosine Ai receptor or antisense to an adenosine Ai receptor mRNA; together with a pharmaceutical carrier. 73. Use of an oligonucleotide (oligo) in the manufacture of a medicament used for reducing the 20 sensitivity to or levels of adenosine, bronchoconstriction, lung inflammation, or allergy or of treating a disease or condition associated with either of them, wherein the oligo is antisense to the initiation codon, the coding region or the 5' and 3' intron-exon junctions of a gene encoding the adenosine Ai receptor or antisense to an adenosine Ai co receptor mRNA; together with a pharmaceutical carrier. o 74. Use according to claim 72 or claim 73, wherein the disease or condition comprises asthma. Use according to any one of claims 72 to 74, S* 30 wherein the subject is a human. 76. Use according to any one of claims 72 to 74, wherein the subject is a non-human mammal. 77. Use according to any one of claims 72 to 76, wherein the oligo is administered in an amount of 0.01 to 150 mg/kg body weight. 78. Use according to claim 77, wherein the oligo is administered in an amount of 1 to 100 mg/kg body weight. H:\Bkrot\Keep\spei\EPI-07 96 b.doc 26/07/00 69 79. Use according to claim 78, wherein the oligo is administered in an amount of 1 to 50 mg/kg body weight. Use according to any one of claims 72 to 79, being a prophylactic or a therapeutic use. 81. A composition according to claim 1 substantially as hereinbefore described with reference to any one of the examples. Dated this 26th day of July 2000 EAST CAROLINA UNIVERSITY By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia S 0 e o S S H:\Bkrot\Keep\speci\EPI-0796bdoc 26/07/00