AU9825398A - Method and reagent for inhibiting human immunodeficiency virus replication - Google Patents

Description

4 S F Ref: 327--472D)

AUSTRALIA

PATENTS ACT 19M FOR A STANDARD

PATENT

ORKA4L namne and Address of Applicant: Rfbozyme Pharmaceuticals. Inc.

2950 Wilderness Place Boulder Colorado 80301 UNITED STATES OF AMERICA Actual Inventor(s): Address for Service: Invention Title: Kenneth G. Draper, Bharat Chowrira, James McSwiggen, Daniel T. Stinchcomb and James D. Thompson Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Method and Reagent for Inhibiting Human ImmW!unodeficiency Virus Replication The following statement is a full description of this Invention, including the best method of performing It known to melus:- Method and Reagent for Inhibiting Human Immunodeficiency Virus Replication Background of the invention This invention relates to the use of ribozyrne?- as inhibitors of human imrmunodeficiency virus (HIV) replication, and in particular. the inhibition of Hreplication. 5-gc, Draper et at., PCT/WG'93/23569 hereby incoriOrtd byrfrence.

Acquired immunodeficiency syndrome (AIDS) is thought to be-caused by infection with the. virus HIy-i At present, it is treated by administration of the drug aizidothyrnidine (AZT), which is thought to slow the pro.-ress of, but not cure, the disezse. AZT resistant strains of HIV-1 are found to develop after a year of treatment.

In some patients AZT has limited efficacy and may be found intolerable. More rccntly, drugs such as dideoxyinosine (DDI) and didecxycytidine (DOG) have been tested as tretnients for AIDS. None of these compounds rcducc the viral load in patients, but they, do treat the disease symptomns.

The following is a discussion of relevant art, none of which is admitted to he prior art to the pending claims. Rossi er al., 8 Aid Reerhai Ijunal eeoynises 183, 1992, pr-ovide a review of the use of ribozymes as anti-HIV-1 therapeutic agents. They state: tmnmnoi Vlftmv An emerging strategy in the treatment of viral infections is the use of antisense DNA or RNA to pair with, and block expressioa of viral transcripts. RNA, in addition to being an informational molecule, can also p s enzymatic activity. Thus, by combining anti-sense and erzymatic functions into a single transcript, it is now possible to design catalytic PNAs, or ribozyTres, which can specifically pair with virtually any viral RNA, and cleave the phosphodiester backbone at a specified location', thereby functionally inactivating the viral RNA.

In carrying out- this .cleavage, the ribozyme is not itself altcred, and is thus capable of recycling and cleaving other molecules, making it a true enzym e. There are several different catalytic motifs which possess enzymatic activity, and each one of these can be inccrporated into an enzymatic antisense with site-specific cleavage capabilities.

Rossi et al. also state that studies have demon- S strated that a hammerhead ribozyme targeted to the gag gene RNA in the vicinity of the translational initiation codon is capable of specifically cleaving that target in a complex milieux of total cellular RNA. With reference to identification of ribozyme targets in HIV-1 they state that mRNAs for the two regulatory proteins tat and rev a-re clearly targets of choice, and that they are examining potential ribozyme cleavage sites in the tat mRNA, as well as in the exon shared by tat and rev. In addition, they state: A rational approach to the problem of target selection i:volves the following criteria.

First, one should select a functionally 5 important target, such as tat, rev, t, psi (packaging site), or the tRNA 1 priming site.

Once a gene or target region has been decided 3 upon, the nucleotide sequence should be assessed for strong conservation of sequence among the Vari~ous isolates. Within these conlserved reglions, the potentaal cleavage sites, preferably GUC. or GUA (others will suf fice, but appitar t-&rbe less efficiently cleaved) shuuld be chosen. The region should be examined i-or potential secondary structures, and then the most promising sites chosen. Finally, before testing the ribozyme in cell culture, it isadvisable to carry out a series of in vitro cleavage reactions (preferably kinetic analyses) using long,:(at least 100 nucleotides in~ length)' substrates to verify that the chosen sites are is truly structirally favorable for cleavage.

[Citation omitted.] Rossi et al. further state that a target which dererves further consideration and testing as a potential ribozyme cleavage site is the viral packaging signal or S0. psi sequence.

.Sioud and Drlic 88 Proc. atl. Acd- Sc. USA 7303, A* 1991 desqcribe ribozymes designed to cleave the integrase gene of HIV. They state that when the ribozyme is transcribed from a plasmid in E. coi ~It leads to de struction 5 of the ipitegrase RNA and complete blockage of integrase Sprotein synthesis. They state that the HIV-l integrase gene may be a useful target for therapeutic ribozymes.

Heidenreich and Eckstvin, 267 Journal of Bioloaical Ihemisr 1.904, 1.992, describe three ribozymes targeted to -:-Qdifferent sites on the long termlal repeat (LTR) RN~A of HIV-l. They also describ-p tjh influence of chemical modifications within the ribozvme on the cleavage of the *.LTR RNA, including 2 '-Fluorocytidiri substitutions and phosphorothioate internucleotidic linkages.

3 weerasinghe et al., 65 Jour anal of iroloc-, 531 1991, describe ribozymes designed against a conserved region within the leader seqruence of RIV-1

RNA;

Chang et al. 2 Clinical BiotecrhnnoloL, 23, !990), describe ribozymes designed to target two different sites in the HIV-l gag gene, and a single site in the viral Sr- LTR region.

-Loren~tzen et al., 5 Virus Genes 17, 1991, describe a ribe zyrne targeted to the virion infectivity factor (vifl of HIV-1..

Sarver et al., 247 Science 1222, 1990, describe ribozymes in the hammerhead family targeted to HIV-12 g ag transcripts. They state that cells challenged with Hiv-1 showed a substantial reduction in the level of HIV-1. gag RVA relative to that in nonribozyrne-expressing cells, and that the reduction in gag R1VA was reflected by a reduction in antigen p24 levels. They state that the results suggest the feasibility of developing ribozynes as rtherapeutic agents against human nathogens such as HqIv-i.

Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filed September 20, 19890, which is a continuation- in-part -of.S. Serial No. 07/247,100, filed September 20, 1988, describe hairpin ribozymes, and provides an example of such a ribotyme apparently specific to the gag gene of HIV-l. Hampel and Tritz, 28 Biochemistry 4929, 1989 and Bampe. et al., 18 Nucleic Acids Research 299, 1990 also describe hairpin catalytic FVRA moels and state that one target site is the tat gene in HIV-1.

Goldberg et WO 91/04319 and Robertson and Goldberg WO 91/04324, describe ribozymes expressed~ within a hepatiti5s delta.-vector and state that the genome of the '3O 0 delta virus aa carry a ribozyme against the en~v or gag mRNA o f HIV. Rossi et al. WO 91/03162, describe chimeric 014 PARIVA catalytic sequences used to cleave 14IV-1 gag trainscript or, the .5 LTR splice site.

Ojwaiq et al, 9 Proc. Natl. Acad. Sci USA 10, 802, ~5 1992 and _YU et al-, 9*0 P Natl. Acad. USA 6340, .1993 describe a hairpin ribozyme allegedly able to inhibit HIV-1 4xpreasion. Joseph and Burkc? 268: i hm 24, 51-5 1993, describe optimization of an anti-HIV hairpin ribozyme. Dropulic et 66 J. Virology 1432. 1992 describe a U5 ribozyme which cleaves at nucleoside +115 in 1-IV-l RNA, SOther related art includes Rossi et U.S. Patents 5,144,019 and 5,149,796; P-ltman et al., U.S. Patent 5,168,053; Zaia et al., 660 Ann. N.Y. Acad. Sci. 9S, 1992; Guatelli et al. 162 J.__Cell BiQchjem. 79, 1992; Jeang et al., 267 J. Biol. Chem. 17891, 199?; Dropulic et al., 66 viral. 1432, 1992; Lisziewicz et International Publication WO 91/10453; International Publicat-ion Wo 91/1S500; Rossi et al., 14A JT. Cell Biochem. D426, 1990; and "The Papovaviridae", Ed. Salzman et al., Vol- 2, Thf Viruses, Plenum Press, N~Y 1987.

Ribozymes are RNA molecules having an enzymatic.

activity which is able to repeatedly cleave other separate RN'A molecules in a nucleotide base sequence specific manner- Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro, Kim et al., 84 Proc. Natl. Acad.

Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; cech, 260 JAI4A 3030, 1988; and Jefferies et 17 Nucleic Acids Research 1371, 1989.

:Ribozymes act by first, binding to a target RNA. Such 5 binding occurs through the target PRNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA w~hich acts to cleave the target RNA. Thus, the ribozyme first recognizes and then. binds tar-get RNA through complementary babe-pairing,,ano once KO bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavace of sujch a target P~x will destroy its ability to direct synthesis of an encoded rtein After a ribozyme has bound and cleaved its- RNA target it is released from that RINA to search for another target and can repeatedly bind and cleave new targets., The enzymatic nature of a ribozynie 3.9 advantageous over other technologies, such as antisense technology 6 (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. in addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of nontargeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing.

Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.

Summary of the invention The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting immunodeficiency virus replication, HIV-1, HIV-2 and related viruses including FIV-1 and SIV-1. Such ribozymes can be used in a method for treatment of diseases caused by these related viruses in man and other animals. The invention also features cleavage of the RNA of these viruses by use of ribozymes. In particular, the ribozyme molecules described are targeted to the LTR, nef, vif, tar and rev viral genes or regions. These genes are known in the art, see, Matsukura et al., 86 Proc. Natl. Acad.

Sci. USA 4244, 1989; Cheng-Mayer et al., 246 Science 1629, 1989; Viscidi et al., 246 Science 1606, 1989; Malim et al., 86 Proc. Natl. Acad. Sci. USA 8222, 1989; Terwilliger et 88 Proc. N'atl. Acad. Scd. USA 10971, 1991; and Bartel et al., 67 Cell 529. 1991, and Figs. 2A and 213.

Thus, in a first aspect, the invention features an enzymatic RNA molecule (or ribozyme) which cleaves IIIV-i RN~A, or its equivalent, regions required for viral repli- Ication, protein synthesis, the vif, nef, tat or rev gene regions, or at structures known to regulate viral gene expression, e.GI., tar, rre or 3'-LTR regions.

By Ilenzynatic RNA molecule' it is meant anr.:Zh-A molecule which has ceipiementarity in a substrate binding region to a specified gene target, and also ha-, an enzymatic activity which is active to specifically cleave RINA in that target. That is, the enzymatic RN~A molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This comple-mentarity functions to allow sutficient hybrid izat ion of the enzymatic F14A molecule to the target RNA to allow the cleavage to occur. One hundred-percent complementarity is preferred, but complementarity as low as 50-75V may also be usflin this invention- By "equivalent-, RNA to HIV-1 is meant to include those naturally occurring RNA molecules associated with immunodefiCiency diseases invarious animals, includ- -ingL humans, felines, and simians. These Viral R1NAs have similar structures and equivalent genes to each other, including the. vif, neff, tat and rev genes.

By ~ee is meant to refer. t hrte protein coding regions of the cognate mRNA, HIV genotme. proviral genome or any regulatory regions in the RNA which reculate synthesis of the protein or stability of the mRl'A.

inl preferred embod iments, the enzymatic Riqh molecule is formed ina hammerhead motif. but may also be tormed in :the motif of a hairpin, hepatitis. delta virus, group) I .:intron or RMaseP-like RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are Sdescribed by Rossi et, al..~e citations above), of Shairpin motifs by 11ampel etal. (see citations above), and an example of the hepatitis delta virus motif is described by PertaadBeen, 31 Biochemistry 1,1992; of the RNaseP motif by Guerrier-Takada, et 35 Ltei38 ~49, in the invention and those skilled in the art will recognize that all that is important In an enzymatic RN~A molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that 8ubstrate binding site which impart an RNA cleavino activitv' to the Molecule.

in particularly preferred embodiments, the R14A which is cleaved in HIV-1 RNA is selected from one or more of the following sequences: Sequence taken from the .HIVPCV12 sequence in the Los Alamos Human Retrovirus and AIDS database. The seauence folded. began *at nucleotide nmber one of the 2.3 kb subgenomic mRZNA. This region includes the coding regions for the vif, v-pr. vpu, tat, rev, and nef gene products.

-Nucleotide Sequence SEQ. ID.NO.

Number 13 AGAA-ACAGUA-ACG~LUU-;AA A I D. i I 108 AGUUUAGUAAJACAC ID.NO.02 121 CCAUAUGUAIJAUUc

DNO

198 UCAGAAGUACACAUC ID.11O.04 228 AGAUUGGUAGUAANA ID. 23-3 AAUAJhCAACAUAUUGG M 06 246 AUUGGGGOICUGCAUA I.O0 2S8 AUACAGGAGAAAGACUGGCAUUJTGGG 1o-110.ae 280 AUCUGGGUCAGGC-AGUCUCCAUA 1D. NO. 0 9 311. A?~.AAAGAGAUAUAGCAOCACAAGU!AGACCCUID .1C -439 UGAACAC rAACAGAGUUUC~i D.!jO* 11 468 AUCLGCCACACLAAACICAAAGqA~ ID -10. 12 601 CACAAUGAAUGGACACUAG 1D.110.13 *644 AAGCUGUUAiGA 1D. 11. 14 683 UAGW, CAAtAUCUAUGAAUpA ID. 800 828 844 875 894 92S 936 988 998 1011 10.'37 1:053 -1126 la 112 1260.

1314 13391 *13 8.3 1 20 1419 14.7 6 1517 .1557 1674 760 3779 1831 1861 1894 1941 r 1966 2065 2188 222f 228:

GCCAJALAUJFAGAA

AUAGGCGUUAC

GAAAUGGAPGCC

AUCCUAGACUAGAGC

AAGUCAG-CCUAAAA

UGUACCAAUUGCUALUGUAAAAAGUG

UUCAlUUGCCPAAG GUUT]GUUUAUACAJAGCCUUAGGCAU7CCU~tGGCAGGC-A

GACAGCGACGAAGAG

AAGACCUCCUiCAAC- GGCAGUC'AGACUCAUCAAGUUTjCUCU

AUCAAAGCAAC

UCCcAUCCCGAGGGA-ZCCCGACAGGCCCGAAGAGCAG'

A

CALUUCG-AUTJAGUGAAZ,

GACGAUU'GCGG-Ar.CCUGUGC.

GGGAAGCCCUcfUAIJGGUC-GAA-uc AGAATJAG-t3CCUG3- UGCCACAGCtIATUArCA ZIACcUAGA AUA L!,GACt'rGCUUGGAGAU UC 'GUCXAAAGJAf, A]AAUGAGACGACA2UGAGCA *GGAGCI4GUAUC73CGA AGAC CUAMAAAArUGG AG CIMILCACA CC-UIC.CUAGAL -GC-ACAAG;3AGGAGGCGGGUGGG ACACCU~l(AGGUACUUUAG-CC:-AUGACt-o.ACAAkG GCkG CUUAGAUCUUACCACUWAAAG-.AG:G GC0GGGGAC\;GG QCUAArUCACUCCCAACGP.

*AGACAGAUAUCCUUG 'IUCUGUGGAUCa-.-CCACA AUUGGCAGACAr-ACA.CCAGGAC UqAGAUAU-CA

AAGCUAGUACCAGUU

GAGAACA4CCAGC110J ACCCUGUGAG CCUGCAUGG.ALUGGAUGA

AGUGGAGGOUMYIACAGCCGC,

CUGCUUTUUUGCCWGU?1C 3UCUGAGCCUGGGAGCUC I U-AAG3CU1GC C ID ID.INC. 2"'L 1D. NO, 22 1ID. 1.1. 23 ID.NO.24 ID .21. 2 62: ID.NO. 26 'ID.-NO.3 ID. NO .31 ID .,NO.32 ID .NO.33 ID.NO.34 1D.N0.36 ID.-NO. 37 ID. NO 38 ID. NO 3 9 ID.N.O.41 ID. NO.42 IM.NO.44 1D.NC. 4 ID. NO.4 6 1D. NO. 7 I D. NO. 419.

19.N1. ~0 ID-NO. 51 ID.NO.52 ID NO.53 ID .NO. 54 rr-+tcl~~. ra*arr 3'LTR;

UGCCUGIAGAUCCUAGAC

AGCAUCCAGC-AAGUCAGCC

nef Qene: 8-23 93-107 214-229 283-297 vif gene: 80-95 167-201 239-253 247-261 286-300 418-432

CAAGUGGUCAAAPI;G

GGAGCAGUAUCUCA

CCNCAGGUACCUJUA

GGGGGACUGGAUGGG

CCAUAUGUAUGUUtJC

AGACUGGUAAUAWAN

-AUCUGGGUCAG-.GAG

AUUTJGGGUCAGGGAG

I'AGGGAGUCUCCAUA

ACACAAGUAGACCCU

AACAAGCUAGGAUCO

ID.NO. 56 ID.N0. 57 ID.NO. 5 6

ID.NO.

ID. NO. Gt ID. N 62 ID.NO. 62 ID. N. 63 ID 64 iD. IM-NO. 66 ID.NO. 67 ID-NO-68 25 In a second related aspect, the invention features a mammalian cell which includes an enzymatic R14A molecule as described above. Preferably, the mammalian cell is a human cell, for example, a T4 lymphocyte having a CD4 receptor molecule on its cell surfacein'a third related aspect, the invention features an expression vector which includes nucleic acid encoding the enzymatj.c RNA molecules described above, located in the vector, 'in a manner which allows expression of that enzymatic- RNA molecule within a mammalian cell.

-In a fourth related aspect, the invention features a method for treatment of human inimuraodeficiency disease by administering to a patient an enzymatic nZA ittolecule which 4:i-iaves HIV-1 RNA or related INA in the vrf, nef, tat or rev gene reg.ions.

In other related aspects, the invention features treatment of cats or simians with ribozymes of this invention. Such ribozymes may be-identical to those able to cleave IV-1I RNA, or may be modified to target analogous locations in FIV and SIV virus pnlqz* :'7 The invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the viral RNA of HIV-1 type virus-infected cells. If desired, such ribozymes can be designed to target equivalent single-stranded DNAs by methods known in the art. The ribozyme molecule is preferably targeted to a highly conserved sequence region of HIV-1 such that all strains of HIV-1 can be treated with a single ribozyme. Such enzymatic RNA molecules can be delivered exogenously or endogenously to infected cells. In the preferred hammerhead motif, the small size (less than 40 nucleotides, preferably between 32 and 36 nucleotides in length) of the molecule allows the cost of treatment to be reduced compared to other ribozyme motifs.

The smallest ribozyme delivered for treatment of HIV infection reported to date (by Rossi et al., 1992, supra) is an in vitro transcript having a length of 142 nucleotides. Synthesis of ribozymes greater than 100 nucleotides in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.

Delivery of ribozymes by expression vectors is primarily feasible using only ex vivo treatments. This limits the S utility of this approach. In this invention, an alternative approach uses smaller ribozyme motifs of the hammerhead structure, shown generally in Fig. 1) and exogenous delivery. The simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure. Thus, unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-ribozyme flanking sequences to interfere with correct folding of the ribozyme structure, or with its complementary binding of the ribozyme to the mRNA target region.

The enzymatic RNA molecules of this invention can be used to treat human immunodeficiency virus infections, including those caused by both HIV-1 and HIV-2. Such treatment can alsobe extended to other related viruses 4*

I

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6

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a j h a :u.

e-

-P

I

r which infect non-human primates including the simian and feline immunodeficiency viruses. Infected animals can be treated at the time of productive infection. This timing of treatment will reduce viral loads in infected cells and disable viral replication in any subsequent rounds of infection. This is possible because the ribozymes disable those structures required for successful initiation of viral protein synthesis.

The targets chosen in the present invention provide a distinct advantage over prior targets since they act not only at the time of viral absorption or reverse transcription during infection, but also in latently infected cells and in virally transformed cells. In addition, viral particles which are released during a first round of infection in the presence of such ribozymes will still be immunogenic by virtue of having their capsids intact.

Thus, one method of this invention allows the creation of defective but immunogenic viral particles, and thus a continued possibility of initiation of an immune response in a treated animal.

In addition, the enzymatic RNA molecules of this invention can be used in vitro in a cell culture infected with HIV-1 viruses, or related viruses, to produce viral particles which have intact capsids and defective genomic 25 RNA. These particles can then be used for instigation of immune responses in a prophylactic manner, or as a treatment of infected animals.

The invention also features immunization preparations formed from defective HIV-1 particles (or related particles) created by a method of this invention, and methods for immunization or vaccination using these defective particles, with DNA or vectors encoding a ribozyme of this invention under the control of a suitable promoter.

i ;i r s :9 -iil^ I- I L_ UI -1 Dianostic uses Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations of viruses within diseased cells. The close relationship between ribozyme activity and the structure of the target

RNA

allows the detection of mutations in any region of the molecule which alters the base-pairing and threedimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to. RNA structure and function in vitro, as well as in cei-'s and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease.

These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent tireatment with combi- S nations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include detection .25 of the presence of mRNA associated with an HIV-1 related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type HIV or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of 35 both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of ri r r cc -3 4i Ii

I

mmm i the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype HIV) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments The drawings will first briefly be described.

Drawings Fig. 1 is a diagrammatic representation of a hammerhead motif ribozyme showing stems I, II and III (marked 30 (II) and (III) respectively) interacting with an HIV- 1 target region. The 5' and 3' ends of both ribozyme and target are shown. Dashes indicate base-paired nucleotides.

Figs. 2A and 2B are diagrammatic representations of the various genes and gene regions in HIV-1 and HIV-2.

S..

*4.

S

S

S

s 2 Figs. 3A 3C are diagrammatic representatiolls of three ribozynes of this invention.

Figs. 4A 4G are diagrammatic representations of chemically modif ied ribozymes of this invention (solid circles indicate modified base Specifically, pig. 4A is unmodified HCH-r37. Figr. 413 is Thio-substituted HcJr37S2,. Fig. 4C is Th O-suIbstituted HCH-r37S4, Figs. 4D- G are Thio Substituted HCH-s37A

D

Fig. 5 is a diagrammatic representation of a chemically modified ribozyme'of this invention (ci'rcled bases are modified with 2 '-O-methyl), specifical.L-y the ribozyme is 21 -O-methyl HCH-037A., Figs. Ga-6f are' graphical representations of ribozvme stability in Vero cell or HeLa. cell extracts from cytoplasm,.membrane, and nucleus.

Figs. 7, 8 and 9 are diagrammatic representations of various hammerhead ribozymes along with data on activity, and liuclease resistance.

Fig. 10 is a graphical representation of activity o f various LTR-targeted ribozymes.

Fi.11 is agraphical representation of activity of *ribozymes of various ,arm lengths.

Fig. 12 isa graphical representation of ribozymes with different sugar modifications.

Fig. 13 is a graphical representation of activity of various TAT-targeted ribozymes.

Fig. 14 is a diagrammatic representation -of a hammerhead ribozyme showing base numbering. Each N and N' can be the same or different.

Fig. 15 is a diagrammatic -representation of two hairpin ribozymes active on HIV

RNA.

:The genome of HIV-j is subject to rapid genetic drift by virtue of its RN~A content and the nature of ezrrors in transcription. Thoge regions (genes) of the genome which are essential for virus replication, however, I--I -e ;Li^ are expected to maintain a constant sequence are conserved) over extensive periods of time. These regions are preferred target sites in this invention since they are more likely to be conserved between different types or strains of immunodeficiency viruses, and thus only one ribozyme is needed to destroy all such viruses. Thus, one ribozyme may be used to target all HIV-1 virus, as well as all HIV-2, SIV and FIV viruses. We have selected several such genes of HIV-1, and examined their nucleotide sequences for the presence of conserved regions which may be cleaved by ribozymes targeted to those regions. Two genes analyzed in detail are the vif and nef genes; the tat, rev and other genes noted above can be analyzed in a manner similar to that described below. Nucleotide sequences were acquired frcm the Los Alamos HIV gene bank.

Ribozymes targeting selected regions of the HIV genome are chosen to cleave the target RNA in a manner which inhibits translation of the RNA. Genes are selected such that inhibition of translation will inhibit viral replication, by inhibiting protein synthesis Selection of effective target sites within these critical S regions of HIV-1 RNA entails testing the accessibility of S.i the target RNA to hybridization with various oligonucleotide probes. These studies can be performed using

RNA

25 probes and assaying accessibility by cleaving the hybrid molecule with RNaseH (see below). Alternatively, such a study can use ribozyme probes designed from secondary structure predictions of the RNAs, and assaying cleavage 4" products by polyacrylamide gel electrophoresis (PAGE), to detect the presence of cleaved and uncleaved molecules The following is but one example of a method by which suitable target sites can be identified and is not limiting in this invention. Generally, the method involves identifying potential cleavage sites for a hammerhead 35 ribozyme, and then testing each of these sites to determine their suitability as targets by ensuring that secondary structure formation is minimal.

s

V

41i The HIV-1 genomic sequences of the Los Alamos data bank were compared in the regions encoding the vif and nef genes. Fifteen putative ribozyme cleavage sites were found to be highly conserved between the 11 strains of virus sequence. These sites represent the preferable sites for hammerhead ribozyme cleavage within these two target PNAs. Two of the nef gene sites overlap regions within the 3'-LTR of the HIV-1 genome, which represents another target of potential therapeutic value. All of the nef targets are present in all known HIV1 mRNAs and may represent targets for cleavage which would disrupt 3' terminal control regions of the mRNA which may be required for efficient translation or export of the uRNAs. In a similar manner, a number of the vif target sites are present in the pol, tat and vpr mRNAs (see Fig. 2).

Short RNA substrates corresponding to each of the vif and nef gene sites were designed. Each substrate was composed of two to three nucleotides at the 5' and 3' ends that would not base pair with a corresponding ribozyme recognition region. The unpaired regions flanked a S central region of 12-14 nucleotides to which complementary S arms in the ribozyme were designed.

The structure of each substrate sequence was S predicted using a standard commercially available PC fold 25 computer program. Sequences which gave a positive free energy of binding were accepted. Sequences which gave a negative free energy were modified by trimming one or two bases from each of the ends. If the modified sequences were still predicted to have a strong secondary structure, '0 the were rejected.

After substrates were chosen, ribozymes were designed to each of the RNA substrates.. Ribozyme folding was also analyzed using PC fold.

-Ribozyme molecules were sought which formed hammer- 35 head motif stem II (see Pig. i) regions and contained flanking arms which were devoid of intramolecular base pairing. Often the ribozymes were modified by trimming a

.I

4..r

S

r ;I r i

;R

p^i; C C I. Ir base fromn the ends-,of the ribozyme, or by introducing additir.nal base pairs :in stem I! to achieve~ the desired f old. Ribozymes with -incorrect folding~ were rejected.

After substrate /ribozyie pairs were found to contain correct. intramolecula'r structures, the molecules were folded together to-predict intermolecular interactions.

A schematic representation -of a ribozyne with its coordinate base pairing tp its cognate target sequence is shown in Fig. 1".

using such analyses, 'the following, predictions of effective target sites in the viE and -e ee ft~ ?V genome, based upon computer generated sequence coinpari sons, were obtained (see Table 1) -The target sequence is listed f irst wit the 5'-most nucleotide number, f or reference. Bases in parentheses are alternative bases-in the conserved patterns.

Table 1 Base numbers .RNA Targret, seauence nef gene .8-23 CAGGUC AAAANG .93-107 G-GAGCAGU A UCtJCCA.

*21472291 CQXcAGG.U A CCUUUA S 28j.-297 -GGGGGAC G GAAGGG AI.SO 3'LTR TARGET)

(U)

430-444 AAGCUAGU ACCAGUUI (ALSO PLTR TARGET) *~vif gene 67 I AGUUUAGU A A;ACAC.

8&-~CCAUAUGU IA UAUUIC1

(G)

-lSd~ -UCAGAAGU A CACAUC 187-201 AGUGUA GJAMNA

(A)

205-220 AUUGGGQG5J C UGCAi0A 19 239-253 AUCUGGGU C AGGGAG

(U)

247-261 CAGGGAGU C UCCAUA 256-300 ACACAAGU A GACCCJ 418-432 AACAAGGU A GGAUCU Those targets thought to be useful as ribozyme targets can. be tested to determine accessibility to nucleic acid probes in a ribonuclease H assay (see below).

This assay provides a quick test of the use of the targct site without requiring synthesis of a ribozyne. it cans be used to screen for sites most suited for ribozvr~e attack.

Synthesis of Ribozymes Ribozytnes useful in this invention can be produced by gene transcription as described by Cech, supra, or by chemical synthesis. Chemical synthesis of RNA is simiilar to that for DNA synthesis. The additional 2'70H group in RNA, however, requires a different protecting group strategy to deal with selective 31-5' internucleotide bondformation, and with RNA susceptibility to degradation in the presence of bases. The recently developed method of RNA synthesis utilizing the t-butyldimethylsilyl group for the protection of the 2' hydroxyl is the most reliable method for synthesis of ribozymes- The method reproducibly yields RNA with the correct 31-51 internucleotide linkages, with average coupling yields in excess of 99V, and requires only a two-step deprotection of the polymer.

A method, based upon H-phosphonate chemistry exhibits a relatively lower coupling efficiency than a method based upon phosphoramidite chemistry. This is a problem for 3 0 synthesis. of DNA. as well. A promising approach to scaleup, of automatic oligonucleotide synthesis has been describe d rec entl.y for the H-phosphonates. A combination of .aproper coupling time an dijnlcapping of *"failure" sequences gave high yields in the synthesis of olioodeoxynucleotides in scales in the-range of 14 gLmoles with as little as 2 eqruivalents of a monomer in the coupling step. Another alternative approach is to use soluble polymeric supports li-- polyethylene glycols), instead of the conventional solid supports. This method can yield short oligonucleot ides- in hundred milligram quantities Der batch utilizing, about 3. equivalents of a monomer in a coupling step- Various modifications to ribozyne structure can be made to en~hance the uf iity ot ribozymes. Such modifications will enhance is. helf -1if e, half-life in -vIritro, stability, and ease cf9jnrdcion of such ribozvmnes to the target site, enhance penetration of cellular membranes, and confer tIe ability to recognize and bind to targeted cells.. ExogenoLS -delivery i ribozyies benefits from chemical modif ication thi L ackbonef by the overall negative chargi the zm molecule. bein g reduced to facilitate f u S o a -Irt7ss the- cell membrane. The present SrtC for redOcing thc bligonucleotide charge include: ,idlf'tcation of- internucleotide linkages by methylphospvnates, use of phosphoramidiLtels, linking oligon-icleot_4e 46 ,to positively cbarged molecules, and creating c packages composed :of oligonucleoatides, and sp,"46-11c receptors or effer-tors for t-rg-eted cells. ExMDe S jf such rnoeif ications Jncjh de' sulfurcontaining ribot*,ymes containing phi~ohioaites and phosphorodithicates a~i in ternucer Timka ges in RNA.

Synthesis of such lfrmdic aoyes is.-achieved by use of the sulfur-tr.-sfer. xeagen' 2 -be-izenedithiol- 3-one !,I-dio:ide., ibzy ,s also contain ribose -modified ribonucle ic-s7 Pyrimidine analogues are prepared from uridine using a procedure employipg diethylamino sulphur-'tritfluoride (DAST) as a starting- reagent.

S 3 Ribozymnes cain also be eitber electrostatically or covalently attached to polymeric cat ions for the pti-pose of reducing charge. The polymer can be attached to the I *ribozyme by simply converting the 3'-end to a ribonucleoside dialdehyde which is obtained by a periodace cleavage of the terminal 2',3'-cis diol system. Depending on the specific requirements for delivery systems, other possible modifications may include different linker arms containing carboxyl, amino or thiol functionalities. Yet further examples include use of methylphosphonates and methyiribose and 51 or 3' capping or blocking with rn.,GpppG or M. 2 27-pppG- For example, a kInased ribozyme is contacted .with guanosine triphosphate and guanyltrailsf erase to add -a r.

3

G

cap to the ribozyme. After such synthesis, the ribozyme can be gel purified using standard procedure. To ensure that the ribozyme has the desired activity, it may be tested with and without the 5' cap using standard procedures to assay both its enzymatic activity and its stability.

Synthetic ribozymes, including those containing various modifiers, can be purified by high pressure liqujid chromatography (HPLC) other liquid chromatography techniques, employing reverse phase columns and anion exchangers on silica and polymeric supports may also be *used.

4.* 4 There follows an example of the synthesis of one .ribo zyme. A solid phase phosphoramidite chernidtry was *employed. Monomers used were 2'-tert-butyl-dimet-hylsilyl cyanoethylphosphoramidites of uridine, kN-benzoyl -cytosine, N-phenoxyacetyl. adenosine, and guanosine (Glen Research, Sterling, VA).

Solid phase synthesis was carried out on either an ABI 394 or 380B DINA/RINA synthesizer using the standard protocol provided with each machine. The only exception was that the coupling step was increased from '10 to 12 minutes- The phosphoramidite concentration was 0.1 M.

Synthesis was done on a 1 pumole scale-using a 1 pmole RNA reaction column (Glen -Research). The average -coupling efficiencies were between-57% and 98t for the 394 model.

1( 9 L 4 22 and between 97% and 99t for the 380B model, as determined by a calorimetric measurement of the released trityl cation.

After synthesis, the blocked ribozymes were cleaved from the solid support (eg. CPG), and the bases and diphosphoester moiety deprotected in a sterile vial by incubation in dry ethanolic ammonia (2 mL) at 550C for 16 hours. The reaction mixture was cooled on dry ice.

Later, the cold licuuid was transferred into a sterile screw cap vial a-nd lyophilized.

To remove the 2'-tert-butyl-dimfethylsily! groubs from the ribozyme, the obtained residue was suspended in tetra-n-butylammonium fluloride in dry THP (TSAF), using a excess of the reagent for every silyl grouP,- for 16 hours at ambient temperature (about 15-2511C1 The reaction was quenched by adding an equal volume of- sterile I M triethylamine acetate, pH 6.5. The sample was cooled and concentrated on a SpeedVac to half the initial volume.

The ribozymes were purified in two steps by HPLC on a C4 300 A5 jim DeltaPak column in an acetcnitrile gradient.

.The first step, or "trityl on step, was a seizarati.on I: of 5'.-DMT-.protected ribozyme(s) from failure sequences lacking a 5'-DMT group. Solvents used for this step were: A (0-1 M triethylammonium acetate, pH 6.8)1 and B (acetonitrile) .Thle elution profile was: 20% B for 10 minutes.

followed by a linear gradient okf 20k B to 5ot B over minutes, 50% B for 10 minutes, a linear gradient of 50!k B S to 100% B over 10 minutes, and a l inear gradient of lo0% R to at a over 10 minutes.

The. second step was a purification of a completely deblocked. ribozyme by a tveatment of trifluoroacetic acid on a.C4 300 A 5 pm DeltaPak column inf an Acetoniztrile gradient. Solvents used for this second step were; 35 (0.J1 M Triethylammoniua acetate, pH4 6.8) and B (80% aceton itrile, 0.1. M triethyl ammuoniumr acetate, pF T he elution prof ile was: 51- B for 5 in~utes. a linear -I--7mrs~ 8rrrrprarrazlarrrraraaaHas~ r gradient of 5% B to 15% B over 60 minutes, 15% B for minutes, and a linear gradient of 15t B to 0% B over minutes.

The fraction containing ribozyme, which is in the triethylammonium salt form, was cooled and lyophilized on a SpeedVac. Solid residue was dissolved in a minimum amount of ethanol and ribozyme in sodium salt form was precipitated by addition of sodium perchlorate in acetone.

or Mg 2 salts can be produced in an equivalent manner.) The ribozyme was collected by centrifugation, washed three times with acetone, and lyophilized.

Expression Vector While synthetic ribozymes are preferred in this invention, those produced by expression vectors can also be used. In designing a suitable ribozyme expression vector the following factors are important to consider.

The final ribozyme must be kept as small as possible to minimize unwanted secondary structure within the ribozyme.

A promoter the human cytomegalovirus immediate 20 early region (HC4V.iel) promoter) should be chosen to be a relatively strong promoter, and expressible both in S vitro and in vivo. Such a promoter should express the ribozyme at a level suitable to effect production of S enough ribozyme to destroy a target RNA, but not at too high a level to prevent other cellular activities from occurring (unless cell death itself is desired).

A hairpin at the 5' end of theribozyme is useful to ensure that the required transcription initiation sequence (GG or GGG or GGGAG) does not bind to some other part of 30 the ribozyme and thus affect regulation of the transcrip- S tion process. The 5' hairpin is also useful to protect S the ribozyme from exonucleases. A selected hairpin S at the end of thi. ribozyme gene is useful since it acts as a transcription termination signal, and as a protection .35 from exonuclease activity. One example of a known termination signal is that present on the T7 RNA polymerase system. This signal is about 30 flucleotides in length. Other 3' bairpins of shorter length can be used to provide good termination anid PI'JA stability. Such hairpins can be inserted within the vector sequences to allow standard ribozymes to be placed in an appropriate orientation and expressed with such sequences attachp-d Poly(A) tails are also useful to protect the 3' end of the ribozyl.~ These can he prov'ided by either including a poly(A) signal site in the expression ve!cto.,- (to signal a cell to add the poly(A) tall in vivo), 6r by. introducing a poly(A) sequence directly inlto the expression vector. In the first approach,,the sional *"i-st be. located to prevent un-,,anted secondary structure formnation~ with other parts, of the ribozyme. In the second approach, the poly stretch m~ay reduce in size over timre wh-en expressed in vivo, and thUs the vector m ay need to bcchecked over time. Care trust.-be takn in addionfa *poly(A) tail which binds poly binding often ak-~kc prevent the ribozyme f ram acting upon their target sequence.

RibozN-me Testingo adOnce the desired riboZyymes are selected, syrt)hesized an purified, they are tested in k inet I-&ic aid other exeri-.

ments to deteltaine their utility.' -Li ean of such a procedure ispr~ovided below.

2.6 Pre~rt~o i6~e 'Crude synthetic rioye p~.350 yjg at t Mefl is p Urified by separationl on a 15V daznaturingPlarb amd gel (0.-75 im.r thick, CIA cmhnq) and visa 4er by U :30 shadowing Once 6xcised e Slesco~~ f3 1en th zibozpre are sokG 4n~g1 tinbf~ LV ECDTAI v C znight with shakiyiq. at T~ Th el car~t atrid Mil Kjand vacu~um dried Tedrjt~i RNA is 01p~ TE (ThIs 1.0 MM'~

D~~

a .6 An aliquot of this solution isdil-it'jd 100-f-old into 1 ml TE, half of which is used to spectropotomet,.ic ally quantitate the ribozyme solution. The conlcentration of this dilute stock is. typically 150.-800 nM._ Purity of the ribozyme is confirmed by the presence of single band on a denaturing polyacrylamidie gel.

A ribozyme may advantageously be synthesized in two or more portions. Each portion of a ribozyme Will generally have only limited or no enzymatic activity, and the activity will inCrease substantially (by at least. 5-10) fold) when all po'rtions are ligated (or otherwisejuxtaposed) together. A: specific example of hammerhead r-ibozyme synthesis is provided below.

-The method- involves synthesis of two (or more) shorter "half' ribozymes and ligaltion of them together usin, RNA ligase. For example, to make a 34 mer ribozytne, two 17 mers are synthesized, one is Phosphorylated, and both are gel purif ied. These purified 17 mars are then annealed to a DNA splint strand complementary to the two 17 mers. This DNA splint has a sequence designed to locate the two 17 -mer ,portions with one end of each adjacent each other. The juxtaposed RNA molecules are then-treated with T4 RNA ligase in-the presence of ATP.

S Alternatively, the DNA splint strand may be oanitted from the ligation reaction if the complementary binding affects favorable ligation of the two RNA molecules. The 34 mer.

RNA so formed is then HFLC purified.

Approximately 1D-30 pnioles of unpurified substrate isA 30 radioactively S' end-labeled with T4 polynucleotide kinase o y 3 PAT.Te entire labeling mix is separated. on a 20% denaturing, polyacry lamide gel and visualized by autoradiography. The full length band is excised and soaked overnight at 40C in 100 y1 of TE (10 mM Tris-HCl pH 7.6, 0.1 mM; EDTA)_ iS -i~ Zlir-r Kinetic Reactions For reactions using short substrates (between 8 and 16 bases) a substrate solution is made IX in assay buffer mM Tris-HC1, pH 7.6; 0.1 mM EDTA, 10 mM MgCl) such that the concentration of substrate is less than 1 nM. A ribozyme solution (typically 20 nM) is made IX in assay buffer and four dilutions are made using IX assay buffer.

Fifteen p1 of each ribozyme dilution 20, 16, 12, 8 and 4 nM) is placed in a separate tube. These tubes and the substrate tube are pre-incubated at 37C for at least five minutes.

The reaction is started by mixing 15 ip of substrate into each ribozyme tube by rapid pipetting (note that final ribozyme concentrations are 10, 8, 6, 4, 2 nM) Five p1 aliquots are removed at 15 or 30 second intervals and quenched with 5 yl stop solution (95t formamide, 20 mM EDTA xylene cyanol, and bromphenol blue dyes). Following the final ribozyme time point, an aliquot of the remaining substrate is removed as a zero ribozyme control.

The samples are separated on either 15% or 20% poly- S acrylamide gels. Each gel is visualized and quantitated with an Ambis beta scanner (Ambis Systems, San Diego, CA).

For the most active ribozymes, kinetic analyses are S. performed in substrate excess to determine K, and K,.

values.

SFor kinetic reactions with long RNA substrates (greater than 15 bases in length) the substrates are S prepared by transcription using T7 RNA polymerase and defined templates containing a T7 promoter, and DNA encoding appropriate nucleotides of the HIV-1 RNA. The substrate solution is made 1X in assay buffer (75 mM Tris- HCl, pH 7.6* 0.1 mM EDTA; 10 mM MgC1 2 and contains 58 nanomolar concentration of the long RNA molecules. The reaction is started by addition of gel purified ribozymes .35 to 1 M concentration. Aliquots are removed at 20, 80 and 100 minutes, then quenched by the addition of S 5 p stop solution. Cleavage products are separated using jv; ii .I-is; 27 denaturing PAGE. The bands are visualized arnd cruantitated with an Arnbis beta scanner.

Kinetic Analysis A simple reaction mechanism for ribozyme -mediated cleavage isk, k 2 R S

.R

where R =ribozyne, S =substrate, and P =products. The boxed step is important only in substrate excess., Because ribozyne concentration is in excess over substrate concentration, the concentration of the ribozvmne-substrate comnplex is constant over time except during the very brief time when the cornplex. is- being initially formed, d[R:Sl 0 dt where t =time, and thus: CR) (RS) (k, 2 The rate of the reaction is the rate of-disappearance of substrate with time: Rate_ ;dt =k 2

(RS)

Substituting these expressions; k, 1/k 2 -dS k') dt or: :3 k 1 k, dt S (k 2 Integrating this expression with respect to time yields: Sk (k, 2

(R)

where So initial substrate. Therefore, a plot of the negative log of fraction substrate uncut versus tme(i minu~tes) yields a straight linwihsoe -SlOPe_ R) =kb.

(kr )I i 28 where k observed rate constant. A plot of slope versus ribozyme concentration yields a straight line with a slope which is: slope which is kat (k 2

K

Using these equations the data obtained from the kinetic experiments provides the necessary information to determine which ribozyme tested is most useful, or active.

Such ribozymes can be selected and tested in in vivo or ex vivo systems.

Liposome Preparation Lipid molecules were dissolved in a volatile organic solvent (CHC13,. methanol, diethylether, ethanol, etc.).

The organic solvent was removed by evaporation. The lipid was hydrated into suspension with 0.1x phosphate buffered saline (PBS), then freeze-thawed 3x using liquid nitrogen and incubation at room temperature. The suspension was extruded sequentially through a 0.4 pm, 0.2 pm and 0.1 pm polycarbonate filters at maximum pressure of 800 psi. The 20 ribozyme was mixed with the extruded liposome suspension and lyophilized to dryness. The lipid/ribozyme powder was rehydrated with water to one-tenth the original volume.

The suspension was diluted to the minimum volume required for extrusion (0.4 ml for 1.5 ml barrel and 1.5ml for 10 ml barrel) with IxPBS and re-extruded through 0.4 im, 0.2 um, 0.1 pm .polycarbonate filters. The liposome entrapped ribozyme was separated from untrapped ribozyme Sby gel filtration chromatography (SEPHAROSE CL-4B, BIOGEL ASM). The liposome extractions were pooled and sterilized by filtration through a 0.2 pm filter. The free ribozyme S was pooled and recovered by ethanol precipitation. The liposome concentration was determined by incorporation of a radioactive lipid. The ribozyme concentration was determined by labeling with Rossi et al., 1992, supra S 35 (and references cited therein), describe other methods suitable for preparation of liposomes.- l i ;;i

P

ic-- 29 In experiments with a liposome formulation composed of a synthetic lipid derivative disteraoyl-phosphatidylethylamidothioacetyl succinimide (DSPE-ATS) co-formulated with dipalmitoylphosphatidyl choline and cholesterol we observed uptake of 100 and 200 nm diameter liposomes with similar kinetics. The larger particles accommodated a larger number of entrapped molecules, or larger molecular weight molecules, such as an expression plasmid. These particles showed a linear relationship between the lipid dose offered and the mean log fluorescence (calcei was used to follow liposome uptake). No cytotoxicity was observed even with a 200 rM dose. These liposomes are particularly useful for delivery to CD4 cell populations.

r c i t r r In Vivo Assay The efficacy of action of a chosen ribozyme may be tested in vivo by use of cell cultures sensitive to HIV-1 or a related virus, using standard procedures. For example, monolayer cultures of HIV-sensitive cells are grown by established procedures. Cells are grown in 6 or *20 96 well tissue culture plates. Prior to infection with HIV, cultures are treated for 3 to 24 hours with ribozymecontaining liposomes. Cells are then rinsed with phosphate buffered saline (PBS) and virus added at a multi- S plicity of 1-100 pfu/cell. After a one-hour adsorption, free virus is rinsed away using PBS, and the cells are treated for three to five days with appropriate liposome preparations. Cells are then re-fed with fresh medium and re-incubated. Virus is harvested from cells into the overlying medium. Cells are broken by three cycles of S30 incubation at -70 0 C and 37 0 C for 30 minutes at each temperature, and viral titers determined by plaque assay using established procedures.

A

1 .7 z&: 4..

4 35 Ribonuclease Protection Assay The accumulation of target mRNA in cells or the cleavage of the RUN by ribozymes or RNaseH (in vitro or in vivo) can be quanatified using an IRNase protection assay.

In t his method, antisense riboprobes are transcribed from template DNA using T7 RNA polymerase

(U.S.

Biochemical) in 20 gl reactions containing iX transcription buf fer (supplied by the manuf acturer), 0. 2 mM ATP, GTP and UTP, 1 UI/il pancreatic RNase inhibitor (Boehringer Mannlheiml Biochemicals) and 200 fICi 3"P-labeled CTP (800 Ci/tnmol, New England Nuclear) for I hour at 370C.

Template DNA is digested with 1 U RNase-free DNaseI Biochemical, Cleveland, OH) at 370C for 15 minute's and unincorporated nucleotides removed by G-50 SEPHADEX spin chromatography.

in a manner similar to the transcription of antisense probe, the target RNA can be transcribed in vitro using a suitable DNA. template. The transcript is purified by standard methods and digested with ribozyme at 37 0

C

according to methods described later.

Alternatively, virus-infected- cells are harvested into 1 ml of PBS, transferred to a 1.5 ml EPPENDORF tube, pelleted for 30 seconds at low speed in a microcentriftige, and lysed in 70 4l of hybridization buffer,(4 M guanidine isothiocyanate, 0. sarcosyl, 25 mM sodium citrate, pH Cell lysate (45 or defined amounts of in vitro :25 transcript (also in hybridization buffer) is then combined of hybridization buffer containing 5 X 10'5 cpm of edch antisense riboprobe in 0.5 ml Eppendorf tubes, over- *laid with 25 Al mineral oil, and hybridization accomplished by :heating overnight at 55 0 C. The hybridization .3 0.3 react ions. are diluted into 0.5 ml RNase solution (20 U/ml P.Nase,2Um ~sT 10 U/mi Rl~ase-free D)NasaIl in 0.4 H ial)-, heated for 30 minutes. at 3711C, and 10 y1 of EDS and 10 Al of: Proteinase K (10 mg/mI) added, followed 1 y an additional 30 minutes incubation at 37CC Hybrids .are partially purified by extraction with 0.5 ml of a 1:1 mixi'r ofpeo/hOoO~auous phases are combined .with 05ml, isopropalol, and R~ase-.resistant hybrids 31 pelleted Lor 10j minutes at room temperature (about 200C) in a Imicrocentrifuge. Pellets are dissolved in 10 gl loading buffer (95k~ formamide, IX TEE, 0.1" bromophenol blue, 0-1k xylene cylanol), heated to 95 0 -C for five mi.nutes, cooled on ice, and analyzed on 4k polyacrylamide/7 M urea. gels under denaturing conditions.

Rihozyme Stability The chosen ribozyme can be tested to determine its stability, and thus its potential utility. Such a-test can also be used to determine the effect of -Qarious chemical modifications addition of a poly(A) tail) on the ribozyme stability and thus aid selection of a more stable ribozyme. For example, a reaction mixture contains 1 to 5 pmoles of S' (kinaEed) and/or.:' labeled ribozyme, 15 fgg of cytosolic extract and -2.5 vM MgCl, in a total volume of 100 ul. .The reaction incubated at 3711C.

Eight pl aliquots are taken at timed lintervals and mixed with a Jlo so i (20 mM EDTA_, 9516 formainide) Samp:les are separated. on a l5V acrylamide sequencing gel, excposed to film, and scanned with an Ambis.

3'-labeled ribozyme can be formed by incorporation 4i of the "P-'labeled cordycepin at the 3' OH using poly(A) pol-,merase.. For example, the poly(A) polymerase reaction S: c-Ontains 40 mM Tris, PH 84 10 m14 MgCl,, 250 mM NaCl, 2.5 mW !4InCl,; 3 Jil P22 cordycepin, 500 Ci/JMN; and 6 units Doly(A) polymerase in a total volume of 50 LIl., The reaction mixture is incubated for 30 minutes at 37 0

C.'

Effet of Idpsoe Sufce Modif ications on Lmphocyte nd _Liposomes containing distearoylphosphartidyl ethanolamidomethyl thioacetate can be prepared. .The thiol group can -be deprotected using hydroxylamine and the reactive thiol can then-be modified with thiol reactive groups to alter-the surf ace -proper ties of the liposomes.. Reaction 'With Nr-ethylmalimide leads to lymphocyte and macrophage 32 uptake. This modification results in liposome uptake by CDC4 and CDC lymphocytes.

Modification with iodoacetamide and iodoacetic acid were tested for uptake. Iodactamide modification showed Sthat the liposomes were toxic in that the lymphocyte cell number decreased with time. However, the acetate uptake was S-fold greater than that observed for succinimide.

Uptake was monitored by both uptake of a 'H-hexaaecyl- J cholesterol ether and an entrapped water soluble fluorophore, calcein. Both confirmed that the liposomes were being taken up by the cells over a 72 hour period.

The uptake was assayed in lymphoblasts which had been stimulated for 72 hours with phytohetnagglutinin. The uptake curve was biphasic in that within the first 8 hours, only cell surface binding was observed whereas after 24 hours, uptake was linear for up to 72 hours.

This was observed for a 200 AM dose. The 100 AM lipid dose showed only cell surface binding.

Effect of Base Substitution Upon Riozme Activity To determine which primary structural characteriistics could change ribozyme cleavage of substrate, minor' base-7

F

changes. can be made in the substrate cle avage regi b ,rrecognized by a specific ribozyme. For example, the substrate. sequences can, be changed at the central "C" nucleotide, changing the cleavage site from a GUC to a GUA a. motif. The values for cleavage using each substrate :are then analyzed.to determine if such a change increases ribozyme-cleavage rates., Similar experiments can be performad to address the effects of cbanainq bases complementary to the ribozyme binding arms. ChI~n rdce to maintain strong binding to the pomplementary substrate *are preferred., Minor changes in nucleotide content can _altet. rihozyme/substrate interact ions -in ways which are *uripted~ct able based upon binding streng!-h alone..

St;-u ttures, in the catalytic core region of the ribozyme h-c~ite trivrial change's in either substrate structure or I 1 1140 a 0.0, MR i MOMME'a 1, W the three dimensional structure of the ribozyme/substrate complex.

To begin optimizing ribozyme design, the cleavage rates of ribozymes contai-ning variead arm lengths,. but targeted to the same length of short RN4A substrate can be tested. minimal arm lengths are required and effective cleavage varies with ribozyine/substrate combinations.

The cleavage activity of selected ribozymes can be assessed using HIV-1-hornologous substrates. The assays are performed in ribozyrne excess and approximate K/~ values obtained. Comparison of values obtained with shoz.t and long substrates indicates utility in vlvo of a ribozyme.

Intracellular-Stability 4of Liposome -delivered Rib-,z:,vries To test the stability of a chosen ribozyme in~vv the following test is useful. Ribozymes are "3ed labeled, entrapped in liposomes and delivered to. HIV-I sensitive cells for three hours. The cells are fracti'Onated and ribozyme is purified by phenol/ch'ror(,f or extraction. Alternatively, cells (x0,T-175 f !ask! are scraped from the surface of the flask and washted twice with cold PBS. The cells are homogenized by douncirng times in 4 ml of TSE (IDk M Tris, pH 7.4, 0.25.M S-acrose, 1 mM EDTA) Nuclei are pelleted at ~oxg forl- m riniutes.

Subcelua oranelles (the membrane ftactiont are pelleted at 200,OO0xg for two hours using an, SW5O -rotor.

The pellet is resuspended in 1 ml of 14 buffer -10.25 M S Sucrose, 50 mM HEPES, pH The suptrnatant contains the cytoplasmic fraction. (in approximat !ly 3.7 The 30 nuclear pellet is resuspended. in I m.1 of G5'i sucrose in TM A SO mM Tris, pH 7.4, 2.5 6M MgCI 1 and bandea ona sucrose step gradient (I ml nuclei in 65k ruczose TM, 1 til6 k sucrose TM, 1 ml 55%, sucr~ose TM, 50t sucrose TM, 300 gI 2S% sucrose TM) for one hour at 37,00OX9 ith an rotor. The nuclear band is harvested and diluted to lot sucrose with TH butffer -Nuclei are pelleted at 37,0 Oxg 77.

34 using an SWG rotor for 15 minutes and the pellet resuspended in 1 ml of TM buffer. Aliquots are size fractionated on denaturing polyacrylamide gels and the intracellular localization determined. By comparison to the migration rate of newly synthesized ribozyme, the various fraction containing intact ribozyme can be determined.

To investigate modif ications which would lengthen the half-life of ribozyme molecules intracellularly, the cells may be fractioned as above and the purity of each. fraction assessed by assaying enzyme ac tivity known to exist in that fraction.

The various cell fractions are frozen at 70 0 C and used to determine relative nuclease. resistances of modif ied. ribozyme molecules., Ribozyme molecules may be synthesized with 5 phosphorothioate or 2' -0-methyl (2 -014e) modif icatiOn8 at each end of the molecule. These molecules and a phosphodiester version of the ribozyme are end-labeled. with 3 2 P and ATP using T4 polynucleotide kinase. Equal concentrations are added to the cell cyto- :plasmic extracts and aliquots of each taken at 10 minute intervals. The samples are size fractionated by denaturing PAGE and relative rates of nuclease resistance analyzed by scanning the gel with an Ambis fl-scanner. The results show whether the ribozymes are digested' by the cytoplasmic extract, and -which versions are relatively more nuclease resistant. .Modified ribozyines generally maintain 80-90% of the catalytic activity of the native ribozyme when short RNA substrates are employed- Unlabeled, 5' end-labeled or 3' end-labeled ribozymescan be used in the assays. These expe rments can Also be performed. with human cell extracts. to v~erify the Observationis.

-IeLa, (CDf4*) and Vero cells are used by many iinvestioscen ftl2ral chemicals in pre-clinical *trials.. Riibozymes and ahtisanse IMAS* depend tupon their nuclease. stability to maintain therapeultic :levels in infected cells- To design chemical modifications which increase-the nuclease stability of ribozymes, we have prepared nuclear, cytoplasmic and membranous lysates from various cell types and Compared the relative specific activity (units of nuclease/ng protein) and cation optima (Mg 2 Mrf 2 Ca*2- or Zn*2-activation) of the nuclease populations within these fractions. Nuclease activities vary significantly among the cell fractions studied (Tables 2 and 3, and Figs. 6a-6f; indicates high nuclease activity, and indicate lower le vels, and i ndicates no ruclease activity as tested by st andard methodology), bvt. the nuclease population present in HeLa cells was dissimilar to that observed in both monocytle and lymphocyte preparations (Table 4, standard units are noted in the-table for comparison only and are not reflective of a specific level of nuclease activity).

Table 2 Relative Activity of Cytoplasimic Extracts Cell Type Units/bug of Divalent cation Protein Requirement Vero Mg*' HeLa .2Zn** Cerviical Epithelial 4.7 Zn'" Monocyte enriched 12.9 none T Lymphocytes 23.5 none Keratinacytes 4-8 n Table 3 Divalent cation eff ect on RNA dearadative activity4In eLa fractions Divalent cation U mMa) 11 fractions 119i Mc1 Cac 1 Mthjc HeLa ce Cyt oplai ZnCl.

+4.4.

membrane.

Muclea~r t 36 Table 4 Divalent cation effect on RNA de~rdatve ctiityin -Vero fractions Divalent cation (I mMj) 'Vero cell fractions Mcrl 2 MnCl, CaCI, ZnCl, Cytoplasmuic Membrane Nuclear HeLa nucleases were. maximally active in the presence of added Zn 2 while added cation did not enhance nik .lease activities in 'onocyte and lymphocyte lysates. The relative nuclease levels in the Monocyte and lymphocyte lysates were 2.2 and 4 times higher, respectively, than in activated HeLa ly~ates.

Various chemical modifications to ribozymes were tested for their ability to increase nuclease activity while maint aining catalytic activity of the ribozyme.

Fig. .7 shows the effects of various modificati~ons upon the catalytic activity of a selected ribozyme. The modifications which decreased activity (1315, 1371, and,128S) were dropped from further analyses. Chemical modifications to ribozymes resulted in -cell-specific patterns of ribozyme stability, anid 21-O-methyl -sugar substitution qave th-e.

best overall enhancement of ribozvme stabiljity -across lysates. Relative resistance of ribozymes to 'diqe.:p-ion by lymphocyte and monocyte cytoplasmic lysate_.r..are showni F: ig. 8. The>digestion rates of the ribozymes are s imiliar when the ribozymes are labeled at either the 51- or 3'ends of the molecule, demonstrating that <the degradation *is -not due to endogenous pohase civt nxhe reports have suggested-hat-modifications to hammerhead motif ribozynes-.in the binding {t~ *and III)-will give munch enhanced protectioui-ft ni digestion. As shown in Fig.' 9, we observe- different 31. results. T~ere-%appear to be 'nucleaee reE 1stant. sites at the positions 2.4, 2.5, 7, 8, 91' 10.4,.and 13. The 2.4, and 10.4 position resistance may be specific to the rib~ozyme tested and more sequences need to be tested.

before a conclusion can be reached concerniiO the nuclease s. sensitivity of these sites,'but the-positions 7, 8, 9 and 13 are conserved nucleotides in this motif and appear to be sites at which nuclease digestion stops, as demonstrated by the appropriate stable fragments produjced in lysate digestion.. experiments. Interestingly, although there seems to be much more nuclease in the* lymphocyte and monocyte lysates, no stable fragments were obsierv,-& when similar experiments were perforrnedusing an activated HeLa.

cytoplasmic lysate. Observations of ribozyme efficacy in HReLa CD4' cells should be re-examined in lymphocytes before 1s the therapeutic relevance can be determinea- Example 1: HIV tat-Ribozymes The 5' exon of cat contains the following potenti-alcleavage sites: 2 GUC sites, 3 6UA sites, 5 AUC sites, 3 UCC sites, and 5 CUC sites. All 18 sites were examined by computer folding and by RNaseH- cleavage assay- :A measure, of the accessibility of each ;site to binding by a 13-mier oligonucleotide was preferred in the.following way: The first 425 nucleotides of the clone V sequence.(this clone was made available by Dr. Rossi and includes the 5' tat exon at nucleotides 151-366) was folded on RNAFOLD 4.0 (a generally available program) and.

examined for the presence of folding domains, L..selfcontained structures closed by a stem.

4. For each potential cleavage site, the domain containing that site w'as folded to confirm that it folded as-in part then the domain was refolded while forcing the cleavage site, and surrounding nucleotides (i1-16 nucleotides in. all) to remain unpaired, The dif ference in 351 these two folding free energies -4as taken as t h* cost of ;1 38 melting out that region for base-pairing by a ribozyme or DNA oligonucleotide.

Ac) The< lengths o f D NA ol igonuc2leot ides. were adjusted to.- :5ve predicted delta-G (binding) of -17 to -18 Skcal/mole. Thus, differences in overall binding energy was predicted to be reflected in the free energy differences calculated in part The calculations are shown in Table Table Total Start Sect 194.

332, 156 211 225 16 28.0 *IL 340 352 240 257 34 6 281 319 322 337.

30 349 SSite GUC 1 (@200) GUC 2 (@338) GIJA 3 (13 t3TJA 4 (@218) GUA 5. (0~233) a.UC 6 (17 AhUC 7 (0-192) AUC 8 (@286) AUC 9 (0347) TJC 10 60) LUC 11 (9248) UIIC 12 (@265) UUC 13 (0354) COC 14 (@288) CUC 15 (@326) CUC 16 (@329) CUC 171 (03441 CUC 18 (@3561, Delta G melting Structure .6 7.5 -4 8.0 6.8 'I4 92.7 +11.9 +13 .8 6-3 I+10.6 Length 12 13 13 16.

134 .12 12 14 Delta G Bindina -17.'S -17.3 -17.7 -17-5 -17.3 -17.7 -17.4 -17.4 *-17.4 -17.4 -17.4 cleaved Delta G Binding 8.6 9.1 -10.2 9.7 12. 6 -12.4 9.9 -15.7 5.7 -6.7 -5.2 -6.A phosphate]I [site refers to nucleotide 5' of 4 4a .4 a p

S

Eighteen DNA oligonucleotides were made to target the 18, target. sites listed in Table 5. fRNasee experiment$ wvere perf ormed using altproximately 100 nM body labeled RN4A

I

is.

39 transcript (clone V) 0.-08 UJ/ 1 1 RJaseH (excess RNaseH) and a 2x dilution of 1 mM, 10 AM or 1 jiM DNA oligonucleot-4de.

The results are shown in Table 5- Eight out of 18 sites had greater than 401f cleavage at 5 AM after 10 minutes in- Three ribozymnes were designed against the three most active seauences (H1332, H337b, 11352, see Table 6).

S

a I S M--c -S S C *t 'S 4 4. a S. P

S

ENERGIES

CLEAVAGE

Oligo same, SequeIce C'3i core Length Binding Melting Net 1mM/301 10~M/1O" 1PM/1CP 11156 GATCTACTGQCT QA1 54-183 360 161 QTCTAGGATCTAC AUC 13 -19.9 5.4 -13.4 12 0 0 12.6 TCCTGGATGCT AUC 11 -18.3 9.4 82 00 191b GraCTGACTTCC Guc 1 -19.6 .6 -19940 H2I1v2 ATTr-GTAC!AAQA GUA 13 -19 a -10 61. 21 0 1422S ACTTTTrACAATAGC GUA 15 -17 6.9 -10.2 47 0 0 H(240 GCAATGOA GCAAC uuC 14, -19.4 12..7 -7,7 25 0 0 1(257 rTGTTATGAAACAAA C OUC- 16 -19 10.7 -7.3 100 0 0 1(290 AGGAGATOCCT AUC 11 -10.2 5.5 -12.7 96 60 0 1(281 ATAGGAGATGCC Cuc 12 -18.5 6.3 -12.2 84 11 0 139GAGGAGOTCTT Cuc 11 -17.4 11.9 -5.5 97 40 0 H4322 CTTGAGGAGGT CtYc 11 -16.9 13.9 -3.1 94 1s. H%32 GTCTGACTGCC GUC 11 -19'.7 8.2 -10.5 94 880 11337b TGATGAGTCTGA CUC 12 -17.6 6.3 -11.3 100 99 14340v2 AACTTGATGAGTC AUC 13 -747.7. -10.3 99 66 0 146ATAGAGAAACTTGA uuC 14 -17.2 9.7 69 100 1349 =TATAGAGAAACT CUC 14 -17.2 10.6 -6.6 55 23 0 145 GTTATAGAGAA -AUC 14 -18. 5 J- 1.7 1-16.8 94 100 6 4a These three ribozymes are shown in Figs. 3A, B3 and c, labeled respectively HDH, HEH and HP11.

Examnje 2: R boZ ies onta n o Th o h s h t TheP~roseofthis example was to evaluate the activity Of riboZymes containing substitution, of thiophosphate for phosphate at some backbone Positions.

Ribozymes to the HIV-j. tat gene were sYnthesized on an AJBI synthesizer using standr 4h~hrmi hm istr h wev r, t s eps where thiophosphate was to be :incorporated in the backbone thec standard oxidati6step (involving Iodine). was replaced. with an oxidation se utiliing te Beacage tep U. trnsfer reagent.

Deprotected and desalted RNA was gel purified,. eluted, kinased .and sequenlced to ensure that. the sequence -was correct. The end-labeled RNA was also treat .ed with which pre -ferentially- Promotes cleavage atPOit2n containing thiophosphate.

Ribozyme activity was tested against cleavageo' sho t 12 uc eot de nd-labeled substrate

RNA.

:s~Substrate concentration was aPproximately I nM; ribozyme concentration wa 5-jao 11M; incubation was at 3 70C in mm Tri s (PH 0.1 mM EDTA, 10 mM MgCl, for 2-40 minutes. Cleavage extents were determined by gel electro.

phoresis (pAGE) followed by quantitation on the AMBIS. The riboZymes are shown in Fis.4 40 Th folowngribozymes showed essential ly 100t activity, r37 (unmodified)' r37s2 (1 thio on 5' arm, 2 on 3 arm) r31s4 (3 thio modIfications on each arm), s37A (3 modfcain modification that.-are fully modified showed some A ecrease in activity, for example, S37C (almost completely :*'modified on substrate binding arms). showed A 3x reduction ain:-activity relative to unmodified, s37D (thio Ihodifica.

tions in- stem. 1, 11 and l) Showed about a 9% redtlction intactivity relative to unmodified.

IA

42 Exam2le 3: Ribozymes were made on an ABI synthesizer using standard phosphoramidite chemistry with phosphoramidite nucleotides used in place of standard nucleotides. Ribozyme activity was determined in the manner described above with PAGE analysis.

Referring to Fig. 5, a ribozym~e containing 2'-Omethyl at all positions that base-pair with substrate (except for the A at the 5' side of stem III) was synthesized. The Kc at/Kmr for the 2'-O-methyl ribozyme was-44 x 106 M-1 min" compared to 41, x 106 m-1 min-' for the unmodified, and 32 x 10' for a thiophosphate modified ribozyme. Thus, the 2'.-O-rnethyl ribozyme retains 1001k.

activity.

V.

Example Targeting the LTR and TAT Reions of HIV-l The f ollowing example extends those provided above to show useful ribozymes targeted to the. LTR. and TAT regions of HIV-1. Details of methodology used herein are provided in Stinchcomb e t al., Methods and Compositions for Treatment of Restenosis and Cancer Using Ribozymes, 17SSN 08/245,466, filed 5/18/94 hereby incorporated by reference herein. Such details are not required to practice the invention. TNmbering .of bases is according to G3enBank Nos. K03455.(IIVHXB2) (numbered from transcription start site).

a at a.

a C 30 a CC a S Screening LTR r i6n fr HR Ribozylle Sies The LTR i s among the most conserved regions within the HIV-l genome. Also, the LTR region is present in all the transcripts generated during-HIV-l life cycle; so, ribozymes that .cleave LTR target s will potentially block HVreplication.

There are 43 potential hammerhead (HI) ribozyme sites within .the, HIV71 LTR.,. Ten hammerhead riboZymes were synthesized based on a) proper folding of the ribozyme-

A.

43 with its target and b) conservation of target sequence among all HIV-1 strains..

RNA Synthesis: Ribozymes with 7/7 binding arms were synthesized S using R1NA phosphoramadite chemistry. Ribozymes were deprotected and purified as described above.

Target RNA used in this study was 613 nt long andj contained cleavage sites for all the 10, HH ribozymes targeted against LTR., A template containing T'7 RNA olymerase promoter upstream of LTR target, sequence, was PCR amplified from an HIV-1 pro-DNTA clone. Other such clones can be readily constructed. Target RNlA was transcribed from thisPCR amplified template using T7 RNAZ.

polymerase. The transcript was internally labeled during transcription by including [&cv-11 CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-1, following transcription at 370C for 2 hours, to digest away the DNA template used in the.

transcription. RNA was precipitated with isopropanol and tOhe pellet was washed two times with 70% ethanol to get rid of salt :and -nucleotides used in the transcription reaction. RNA: i s resuspended in DEPC-treated water and stored at*I4W1.

Ribozvme-:glqayg~ eact ions Reactions were carried. out under ribozyme excess (k*/.)conditio:ns (Herschlag and Cech U990) M.Lt~ 1.,10159 10111) .Bri;efly,. 1,000 nM ribozyme and 10 NM I iternally labeled target RNA were denatured separately by heating to '900C for2 min in th rsnce of 50 M4 Tris.HC1 1 PH{ 7.5S and 2:0 mM Mgcl-. The RNAs were renatured by cooling..to 37 0 C for 10-20,min. Cleavage reaction was initiated by mixing the ribozyme and target'RNA at 37 0

C.

A3.iquots of 5 jzl were-taken at regular inttrvals ol time and the reacti6n was quenched: by: addincg equal- volume of

S

a.

DI

stop buffer and freezing on dry ice. The samples were .resolved on sequencing gel.

Ae shown in Fig. 10, of the ten ribozymes that were tested individually, only four HHi ribozvmes (568, 561, £31, 696.) cleaved the LTR RNA. Other target sites appear to be inaccessible to ribozyne binding and cleavage. Site 56S is also referred to as site 115 and has been targeted for hammerhead ribozvme cleavageby Drouplic etal,, 66 J.

'Jirol. 1432-1441, 1992 an d Heidenreich and Ecksteid, .267 J. Biol. Chem. 1904-1909, 1992.

Since *S68 HII ribozyne was cleaving its target to a greater extent than the others, we were interested in optimizing the length of binding arms of this HR! ribozyme.

As shown in Fig. 11, the -reof ribozyme, cleavage increased significantly when tb r :Isngth of the binding armn wasincreased from 12 to 14 ba, pairs (total). There was no significant improvemnent in the activity of ribozymes with binding-arms longer than 14 base pairs.

'Zo Chemical modification of 1i{B ribozymes targeted to a *specific site. can-significantly improve -the stability of :.the ribozyme in human serum. Further,, these modifications &o not seem to have any significant effect on the catalytic activ ity of the. ribozyme. All the 2' hydroxyl1 -groups, within the ribozyne, with. the, exception of positions U14, G54 A6, U17, G8, G12 and A15-1 (using standard nomenclatures See Fig. were modif ied with 2'-Q-methyl *:groups. The .21 hydroxyl groups at .U4 and M1 were modifie wtheithr ain,2-C-allyl, 2'-O-methyl or 21ira-tlouro. See Usman et al., 2'-Deoxv 2'- .~alkylnucleotide containing Nucleic Acid, USSN 06/218,9-4, f iled March. Z9, 1994, hereby incorporated by ref erence herein.

Ref erring to Pig. 12, the 5 68 oiH -ri-bozyme could be -stabilized by chemical modification.-Of the sugar rnoeity of various bases (&imjlar. to the ones listed-above). The 56$

*IJ

f

I

S 3 HR. ribozyme was extensively modified with one of the following compounds: 2'flouro, 2'amino at the 07 Position, araflouro at U4 position, 2'amino at 04 and U7 pogitions, c-allyl at U4 position. None of the above mnodifications had any deleterious effect on the ribozyme activity.

Transfection of cells with 568 HH ribozyme (with U4 and U7 positions containing 2'amino modifications:! blocks the replication of HIV-1 replication.

Screening TAT region for RRibozvme Sites: A region of the TAT mRNA is present in the maj-ority of the transcripts generated during HIV-2. life cycle. So., ribozymes that cleave TAT targets will potentially block HIly replication.

There are 54 potential HH ribozyme site within the HIV-l TAT regions (between 5776 nt and.6044. nt). Nine hammerhead ribozymes were synthesized based on, a) proper folding of the ribozyme with its target and b) coriservation of target sequence among all HIV-,l strains.

RNA synthesis and ribozyme cleavage reactions were io carried out as described above. Target RIIA used i n this study was 422 nt long.

As shown in Fig- 13, five sites (5841, 5869- 5878, 5966, 5969) are cleav%.ed more readily than *teothers'.

None of these hammerhead sites have previously been ,2S targeted.

3D 4~ V V

S

Screening HIV-1 qenome for Hairpin Pijbozvm) ie± Refer-ing to Table VIII and Fig- 5.Ueeae2 potential hairpin (HP) ribozyme sites in the IV-ge'o.

Ribozymes shown in the table were syntnIesized and te~ted, Modifications to various regions of the haip .srutue can-be. made without deleterious effect., in. thoset~iragetted. to positions 565 And 4398--two extra bas~es can be, insertedin place. of GA {3rd ol.) OGGAi and GCAQ to GCAGUC repectively, 4- 46 Site 565 within the LTR region (Qiwang et al., (1992) PNAS. USA. 89, 10802-10B06; Yu et (1993) PNAS.

USA. 90, 6340-6344) and 4398 within the POL region of the HIV-1 genome (Joseph and Burke (1993) J. Biol. Chein. 268, 24515-?.4518).have been shown to be accessible to HP nibozyme biading and cleavage. We have also found that 11P.

ribozymes targeted towards.565 and 4398 sites are active,.

The best HH and HP ribozvnes are shown in Table VII, with their associated c leavage and target sites- Administration of Ribozyone Selected ribozynes can be administered prophylactically, or to HIV-1 infected patients, by exogenous delivery of the ribozyme to an infected tissue. by means of an appropriate delivery vehicle,. a liposone, a controlled release vehicle;' by us of' iontophoresis.

electroporation or ior paired molecules, or covalently attached *adducts, and other' pharmacologically apprved methods of delivery. Roijtes of administration include intramuscular, aerosol, oral (tablet or pill formt, topical. systemic, ocular, intraperitoneal and/or intra- *thecal. Expression vectors for immunization with ribozymes and/or delivery of ribozymes are also suitable.

The specific delivery route of any selected ribozyme will depend on the use of the ribozyme. Generally, a specific delivery program for each ribozyme will focus on unmodif ied ribozyme uptake with regard to intracellular localization. -followed by demonstrat ion _of.e f ficacy.

Atraively, delivery to these same cellIIs in an ogno Stissue ofian animal can be pursued' Uptake studies will include, uptake assays to ,evaluate cellular ribozyme uptake, regardless of the delivtery vehicle pr strategy.

Such assays wil). also .determin 7ihe iniacellular localiZationI 6Z the riboryme fol 6*in9 'uptake,, ultimately estabIishingo the. requirementi for maintenance of steadystata cowentrations, wiLthin 'the cellular compartment cnininq tile targe sIzqncle (nucleus nlrcyols) et sbe. adlor ytopasm) 47 .Efficacy and cytotoxicity can 'hen be tested. Toxicity will not only include cell viability but also cell f unct ion.

Some methods of delivery that may be used include: a. encapsulation in liposomes, b. transduction by retroviral vrectors, c. conjugation with cholesterol, d. localization to nuclear compartment utilizing antigen binding or nuclear targeting site found on most snRNAs or nuclear proteins, e. neutralization of charge of ribozyne by. -using nucleotide derivatives, and f. use of blood stem cells to distribute ribozymes throughout the body.

ISAt. least three types of delivery strategies are useful in the present invention 1 including: ribozyrre modifications, particle carrier drug delivery vehicles, and retroviral expression vectors. Unmodified ribozymes, like most small molecules, are taken up by cells, albeit slowly- To enhance cellular uptake, the ribozyme may Lre modified essentially at ranldom, in. ways which reduce its charge but maintains specific functional groups- This retults in a molecule which is able to diffuse' across the cell,"membrane, thus removing the permeability barrier.

Modification of ribozymnes to- reduce charge is Just.

one approach to enhance the cellular uptake of theseliarger-.molecules. The random approach, however, is not.

advisable since ribozymes are structurally and functionally more omlxthan small drug molecules. The structural- requirements 'necessary to maintain ribozyme catalytic activity are well understood by those in the art.. These requirements are taken into consideration when designing modifications to enhac celua eiey The modifications are also designed. to reduce sixaceptibility to nuxclease degradation. -Both of tbese characteristics should' greatly improve. the ef ficacy of the ribozymei.

cellrupaecnbe.- increased by iseveral orders of Cellr ae :a 48 magnitude withou t having to alter the phosphodiester linkages necessary for ribozyme cleavage activity, Chemical modifications of the phosphate backbone will reduce the negative charge allowing free diffusion across the membrane. This principle has been successfully demonstrated for antisense DNA technology. The similarities in -chemical composition betwnen DNA and RNA make this a feesible approach. In the body, maintenance of an external concentration will be necessary to drive the diffusion of the modif ied ribozyme into the cells of the tissue.

Administration routes which allow the diseased tissue to be exposed to a transient high concentration of the drug, which is. slowly dissipated by systemic adsorption are preferred. Intravenous administration with a drug carrier designed to increase the circulation half-life of the ribozyme can be used. *The size and composition of the drug carrier restricts rapid clearance from the blood stream.- The carrier,,made to accumulate at the site of ifection, can protect the ribozyme from degradative processes.

*Drug delivery vehicles are. effective. for both systemic and topical administration. They can be designed to septre as a slow release reservo~ir, or to deliver their contents directly to the target cell. An advantage of using direct. delivery drug vehicles is that multiple molecules *are delivered per uptake. Such vehicles have been shown to increase the circulation half-life of drugs which would otherwise be rapidly cleared from the blood stream.. Some examples of. such specialized drug delivery O vehicles which fall into this category are -liposomes, hydrogels, cyclodextrns, biodegradable nanocaosules, and bioadhesive microspheres.

~'rm tiscategory of delivery systems, Ilipos omag. are pref6rremesA increase intraelular stabi-Ljy inres e uptake efficiency and improve biaolical

QI

Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. They have an ihternal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter. Several studies have shown that liposomes can deliver RNA to cells and that the RNA remains biologically active.

For example, a liposome delivery vehicle originally designed as a research tool, Lipofectin, has been shown to deliver intact mRNA molecules to cells yielding production of the corresponding protein. In another study, an-antibody targeted liposome delivery system containing an RNA molecule 3,500 nucleotides in length and antisense to a structural protein of HIV, inhibited virus proliferation in a sequence specific manner. Not only did the antibody target the liposomes to the infected cells, but it also triggered the internalization of the liposomes by the infected cells. Triggering the endocytosis is useful for viral inhibition. Finally, liposome delivered synthetic .0 ribozymes have been shown to concentrate in the nucleus of H9 (an example of an HIV-sensitive cell) cells and are functional as evidenced by their intracellular cleavage of the sequence. Liposome delivery to other cell types using S smaller ribozymes (less than 142 nucleotides in length) exhibit different intracellular localizations.

Liposomes offer several advantages: They are nontoxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to 0 o tissues. FiSiy, cost effective manufacture of liposomebased ha uceuticals, either in a liquid suspension or Slyopt ~d product, has demonstrated the viability of Sthis technology as an acceptable drug delivery system.

O' ther controlled release drug delivery systems, such as nonoparticles and hydrogels may be potential delivery vehicles for a ribozyme. These carriers have been developed for chemotherapeutic agents and protein-based -a ~C I -Ir pharmaceuticals, and consequently, can be adapted for rihozyne delivery.

Topical ad-ministration of ribozymes is advantageoils since it allows localized concentration at the site of administration with rinimal systemic adsorption- This simplifies the delivery strategy of the ribozyme to the disease site and reduces the extent of toxicological, characterization. Furthermore, the amount of material to be applied is far less than that required for other 10 admninistration routes. Effective delivery requires the rib~ozyme to diffuse into the infected cells. Chemical modification of the ribozyme to neutralize negative charge may be all that is required for penetration., However, in the event that charge neutralization is insufficient, the modif ied ribozyme can be co-formulated with permeability enhancers, such as Azone or oleic acid, in a liposone- The liposomes can either represent a slow release presentation vehicle in which the modified ribozyme and permeability enhancer transfer .from the liposome into the '20 infected cell, or the liposome phospholipids can participate directly with the modified ribozyme and permeability enhancer in facilitating cellular delivery. In sonie cases, both the ribozyme and permeability enhancer can be formulated' into a suppository formulation *for slow release.

*Ribozyies may also. be systemically administered.- Sytei absrpio refers toteAcmlto of dusin the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic include: intravenous, subcutaneous, intraperitoneal, intranaual, intrathecal And ophthalmic. Each of these administration routes expose the ri4bozyme to an accessible diseased tissue. Subcutaneous administration drains into a localized lymph node which proceeds through the lymphatic network into the. circulation. The rate of entry ihto the ,circulativn has been shown to be a function of tnolecu.lar weight or size. Te u.eo lipoosome or* a.

51 other drug carrier localizes the ribozymfe at .the lymph node. The ribozyme can be modified to diffuse into the cell, or the liposome can directly participate in the delivery of either the unmodified or modified ribozyme to the cell. This method is partiCularly useful for treating AIDS using anti-HIV ribozymes of this invention.

Also preferred in AIDS therapy is the -use of a liposome formulation which can deliver oligonucleotides to lymphocytes and macrophageS. This oligoflucileotide delivery system inhibits i4IV proliferation il-, inftected primary immune cells. Whole blood studies show that the formulation is taken up by 90V of the lymphocytes after 8 hours at 370C. Preliminlary biodistribution and pharmaco-.

kinetic studies yielded 70;; of the injected dose/gm of tissue in the spleen after one. hour following intcravenous administration. This for-mulation off ers an excellent delivery vehicle for anti-AIDS ribozymes for two reasons.

First, *r-helper lymphocytes and macrophages are the S primary cells infected by the virus, and second,_a subcutaneous administration delivers the ribozymes to the resident i1IV-irfected lymphocytes and macrophages in the *lymph node. The. liposomes then exit the lymphatic system, enter the circulatione and accumulate in the spleen, where the. ribozyme is delivered to the resident lymphocytes and macrophages.

**.Intraperitaneal administration also leads to entry into the circulation, with once again, the molecular weight or size of th iozyme-delivery vehicle complex con~ 0 trollinlg ther ate of entry .*i'43 Liposomes injected intravenlously show accumulation in I, te liver, lung and spleen, The composition and size can be adjusted sota-hsacuuainrepresents 30N to of the injected dose., The remaining dose c' :cular-es in the blood streamU for up to.24 hour,-.

The hoelmethod ofdeivery should, result in cytoplaatic accumulation, if the afflicted cells and.-= molecules should have some huclease-Tesistance folO ttimo: 52 dosing. Nuclear delivery may be used but is less preferable.- Most preferred delivery methods include liposornes ilO1-400 rim), hydrogels, controlled-release polymiers, tni:roi~njectiol or electroporation (for ex vi vo treatments) and other pharmaceutically applicable vehicles. T'he dosage will depend upon the disease indication- and the route of-.admi-istration but should be between 100-200 mg/kg of body vweight,/d-ay-.. The duration of treatment will extend through the couarse of the disease symptoms, usually at least 14-16 days and possibly continuously. Multiple daily doses are anticipated for topical applications, ocular applications- and vaginal applications. The number of doses will depend upon disease delivery vehicle and efficacy data from clinical trials.

Establishment of therapeutic levels. of ribozyme within the cell is dependent upon. the rate of uptake and degradation. Decreasing the degree of degradation will prolon the intracellular h?;-lif of the rOYme..

Thus, chemically-mdfe q.6ozyde f 4 h~n~fcation of. the phosphate backb one, or 'capp-rI. f th 5' a nd 31 ends of. the rboy i ith nucleotide an~logue. may require different dosaging. *.Descriptions of uS -a fuI systems are provided in the art cited.-Above,~ all of which hereby;,incorporated by -'.,srence herein.

Th lie ioymes a-e 4so useful as diagnosti topls :to specifically orn-pecifically detect the prusence of a. target RNA in a rne.That;As the target RNA, if present: in the. saT~p16., will .be *.specifically :0cleaved, *by the ibozysf,, an-i! thus can be readily: and specifically detected an smfaller RNA species. The presenc -o sh sallerFW. pecies is indicative of the Presence. of the taiet R~ iJ..n the sampe Othet embadin, et~- within the following- &iaims.

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I

two 08/103,243.

AU9ust 6, 19931 4* 4 .4 APPLICATION NUMBER: 07/882,886, FILING DATE: May 14, 1992 (viii) ATTORNEY/AGENT INFORMATION: NAME: Warburg, Richa REGISTRATION NUMBER: 32,327 (C)-REFERENCE/DOCKET NUMBER: 206/116 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (213) 489-16 TELEFAX: (213) 955-04 .0 TELEX: 67-3510 INFORMIATION FOP, SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: (B)TYPE: nucleic LS 5 STR.ANDEDNESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AGAAGAAAAG CAAAGAUCAU UAGGGAUUAU GGAAAAChGA ()INFORMATION FOR. S3Q ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 1 TYPE: nuclei STR.ANDEDNESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2: AGUUUAGUAA AACAC INFORMAkTION FOR SEQ ID NO: 3! UY .SEQUENCE CHARACTERISTICS: LENGTH: 3p(B) TYPE: nuclei _STRANDEDNESS single TOPOLOGY. linear (i SEQUENCE DESCRIPTION: SEQ ID NO: 3; CCAUJAUGUATU AUJUUC .15 INFO RKwATIONk FOR- SEQ XDNO: 4: SEQUENCE 0iARACTERISTICS: LENGTH- 1 rd 00o acid acid .4 .4 i

V

cacid

I

TYPE:

STRANDEDNESS:

TOPOLOGY:

(ii) SEQUENCE DESCRIPTION: SEQ ID UCAGAAGUAC ACAUC INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

(ii) SEQUENCE DESCRIPTION: .SEQ ID AGAUUGGUAG UAANA INFORM-IATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

SEQUENCE DESCRIPTION: SEQ ID AAUAACAACA UAVUGG INFORMATION FOR SEQ ID NO: 7: Wi SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANqDEDNESS:

TOPOLOGY-

(ii) SEQUENCE DESCRIPTION: SEQ ID AUUGGGGUCU GCAUA INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS-

LENGTH:

TYPE:

STRANDEMNESS.

TOPOLOGY*

(ii) -SEQUENCE DESCRIPTION.- -SEQ ID, AUACAGGAGA AAGAGACUGG CAUUUG nucleic acid single l inear NO: 4:- 1s nucleic acid Sirngle linear~ NO: nucleic acid single 1 inear NO: 6: nucleic acid s inale linear NO: 7: 18 nucleic acid single linear.

NO: 8:

I

0. 20 .3 C* INFORMATION FOR SEQ ID NO: 9: SEQUENCE

CHARACTERISTICS:

LE1NGTH: 23 TYPE: nucleic z STRANDEDNESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AUCUGGGUCA GGGAGtJCUCC AUA 23 INFORMATION FOR SEQ ID NO: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: TYPE; nucleic ()STRANDEDNESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: AAAAAGAGAU AUAGCACACA AGUAGACCCU INFORMATION FOR SEQ.ID NO: 11: SEQUENCE

CHARACTERISTICS:

LENGTH: 42 TYPE: nucleic STRANDEDNESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 11: UGAAUAflCAA GCAGGAcAUA AcAAGGuAGG kUCUCUACAA UA 42 ()INFORMATION FOR SEQ ID NO: 12: SEQUENCE

CRAPRACTERISTICS:

LENGTH, 44 T'YPE: nucleic STRANDEDNESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO;. 12 AUACUUGGCA CUAGCAGCAU UAAUAACACC AAAAAAGAUA AAGC 4 ()INFORMATION FOR SEQ ID NO: -13: (4SEQUENCE

CHARACTERISTICS;

(A)"LENGTH: .19 TYPE: nuclei STRANDEDNESS: single ~cid acid acid acid c acid -em 120

V..

s

TOPOLOGY:

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: CACAAUGAAU GG~ACUAGl 19 INFORMATION FOR SEQ ID NO: 14: Wi SEQUENCE

CHARACTERISTICS:

LENIGTH: 1 TYPE-. nlu STRANDEDNESS: Si Top OLOGY: ii (ii) SEQUENCE DESCRIPTION: SEQ ID NO: AAGCtJGUUAG A 1 INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:.

LENGTH: TYPE: riuc (IC) STR.ANDEDNESS: i TOPOLOGY: h (iSEQUENCE DESCRIPTION: SEQ ID 1NO: UAGGGCAACA UAUCUTtGAA IACUUA ()INFORMATIONFOR SEQ ID NO: 16: Wi SEQUENCE

CHARACTERISTICS;

LENGTH: 13 TYPE: nuc STRANDEDNESS:- sin TOPOLOGY: IirA (iSEQUENCE DESCRIPTION: SEQ 'DN:16 GCCAUAAUAA GAA 13 INFORMATION FOR SEQ ID .NO: 17: ()SEQUENCE

CHARACTERISTICS:

LENGTH: 1 ()TYPE: nluc STRANDEDNESS: sin( .(I)TOPOLOGY: lr (ii) SEQUENCE DESCRIPTION: SEQ ID NO: AUAGdCGuUA 'C IFORMAION FOR SEQIDNO 18 U1) SkQMECE

QIARACTERISTICS.

.near 13: .cleic acid ngle near 14: lic'acid igle iear leic acid gie ear leic acid ;l e ,ar 17:

I

I-

LEN'"TH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

SEQUENCE DESCRIPTION: SEQ ID GAAAUGGAGC C INFORMATION FOR SEQ ID NO. IS: SEQUENCE CHARACTERISTICS:

'LENGTH:

TYPE:

STRANIJEDNESS:_

TOPOLOGY:

(ii) SEQUENCE DESCRIPTION: -SEQ ID, AUCCtJAGACU AGAC INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STR.ANDEDNESS:

TOPOLOGY.

(ii) SEQUENCE DESCRIPTION: SEQ ID AAGUCAGCCU AAAA INFORIATION FOR SEQ ID NO: 21: Mi SEQUENCE CHARACTERISTICS;

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

(ii):SEQUENCE DESCRIPTION: SEQ ID UGUACCAAUEJ GCUAUIJGUAA.AAAGUG INFORMATION FOR SEQ ID NO: 22: (ii SEQUENCE CMARACTERISTICS-

LENGTH.

TYPE:

STRANDEPNESS:.

.1D) TOPOLOGY: iiSEQUEN-CE DESCRIPTION: SEQ. ID 11 nucleic acid single 1linear NO: 1S: n ucleic, acid single' linear NO: 19: 14 nucleic acid single linear NO0: 14 26 nucleic acid sin gle.

linear NO: 21: 26 12 nucleic acid single linear NO: 1.22- *9t~8 1~ tJUCAUUGCCA AG2 INFORMATION FOR SEQ ID NO: 23: SEQUENCE

CHRACERISTICS:

LENGTH:4 TYPE: nucleic acid STRANDEDI ErSS: single TOPOLOGY:. linear (ii) SEQUJENCE DESCRI)PTION- SEQ ID, NO: 23: GUU UGUU.CA tAACAAAAGC CUUAGGCAUC UCCUAUCGC-A GGAA 44 INFORMATION FOR SEQ ID NO: 24: ()SEQUENCE CHIL;AcT3

RIS"ZICS:

LZ-NGTI: 1 TYPE: -nucleic acid (IC) STRANDIRDNSS: 7singl~e TOPOLOGY: -linear (ii) SEQUENCE DESCRIPTION:. .0EQ ID10f: 24: GACAGCGACG.AAGAG i INFORMATION FOR SEQ ID NO: 2: (iJ) SEQUENCE

CHARACTERISTJS

LENGTH: 14 TYPE: nucleic acid STR.ANDEDNESS. sn1 TOPOLOGY, in~ .(i)SEQUENCE DESCRPTION SEQ I D NO:2 2 5~ AAGACCUCCU CAAG2.

()INFORMATION FOR SEQ ID NO: 26: SEQUENCE*

CHRACTRXSTICS:

C(A) L N T e26.

Li) ns-.:fucleic acid STRAflEMiMSS: single TOPOLOGY:'- 'near SEQUENCE DECItO: ID No:. GGCAGUCAGA CUCALUCAAGU tlUCUCU 26 *Ot: INtFORMATION FoR S ID .ND i}SE-QUENCE

CAATRSIS

fulueie, acid~

I

C) STRANDtflNESS: NJO: 2'7: ToQo -0111Y.

fili) QRTJMt'C8 DESCRIPTION: SEQ ID AUCUAS--AP -C INFORMATION FOR SEQ ID NO: 28:..

SZQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STR.AhDEDNqESS,:

TOPOLOGY:

(ii) SEQUENCE DESCRIPTION: SEQ ID UCCCAAJUCCC GAGG;GGACCC GACAGGCCCG AGAUG INFORMATION FOR SEQ ID NO: .29: Ui SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY.

(i)SEQUENCE DESCRIPTION: SEQ IM *0CAUUCGAUUA GUGAA

ENGTH.

TYPE:

STRANDE01NESS: ID) TOPOLOGY, (ii) SEQUENCE DESCRIPTION: SEQ 10 GGACGAJCUG CGGAGCCUGU GC IFORMATION FOR SEQ ID NO: 31: SEQUZINCE CHARtACTERISTICS:

L.ENGTH!

TYPE:

STRANDEDNESS-

C)TOPOLOGY:

EQUENCE, DESCRI PT ION: SEQ ID -GOG3AGcC CP.AAUAVTJGr- UGGAAUCOC awORa~xo~ PQ.S~QID 10; 2% t, Pd 44 nucleic acid single linear* NO:28 AGAA 44 nucleic acid.

single linear NO0 29: 22 nucleic acid single linear NO; 22 29 hiucleic acid aingle linear NO 3,L: 29

V.

I

Wi SEQUENCE -HARACTERISTICS: LENG-1H: 12 TYPE: nuclei STRANDEDNESS: singi TOPOLOGY: iinea {ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3 AGAAUANGOGC UG 1 INPORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH:1 (B.1 TYPE: nucle STRANDEDNESS: singi1 TOPOLOGY: linea (ii) SEQUENCE DESCRIPTION: SEQ ID NO:.

UGCCACAGCU AUAGCA 16 INFORMATION FOR SEQ ID NO: 34:- SEQUENCE CHARACTERISTICS: LENGTH: 23 TYPE: nucle STRANDEDNESS: in TOPOLOGY: n (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 34: AAGUAGUJACA AGAAGCUUAU AGA 23 INFOR MTION FOR SIEQ ID NO: 4-2s Wi SEQUENCE CHARACTERISTICS: LENGTH: 34 (BI, TYPE: nuci STRANDEDtIESS: sing TOPOLOGY: line 39 (ii) SEQUNCE DESCRIPTYC0:; SEQ ID NO: UACCUAGAAG AA.UAAGACAG GC-UTGGAAA GGAJ 34 ()INFORMATION FOR SEQ ID NO: 36: C)SEQUENCE

CHARACTERISTICS:

5(B) TYPE:- nuc) STRANDEDNESS- i 'rOPOLOQ: lIin ~c acid e f 2: ic acid er Ti i zid ei.c acid le ar ~4 I .7 Leic acid ear A A MC SEQUENCE DESCRIPTION: SEQ lID NO: 36: tJGGUCAiAAAA GTJAG 14 INFORMATION FOR SEQ ID NO: 37: Wi SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iiJ) SEQUENCE DESCRIPTION: SEQ ID NO: 37: AAGAAUGAGA CGAGCUGAGC CA 22 M2 INFORMIATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRAN'DEONESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GGAGCAGUAU CEJCGA INFORMATION FOR SEQ ID N 39; Wi SEQUENCE CHARACTERISTICS: LENGTH: 28 TYPE: nucleic acid STRANIDEDNESS: single TOPOLOGY: lineax- (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 39., AGACMUAGILN AAACAUGGAG CAAUCACA INFORMATION FOR SED 1D NO: Wi SEQUENCE CHARACTERISTICS: LENGTH:. 34 IS() TYPE: nucleic aci d STRAN2DEDNESS: snl TOPOLOGY:l-na SEQUENCE DESCRIPTION: SEQ 1D No.. :0 ccuGCUAGA AGCACAAGAG GKAGGAQAhGG tJGGGy10 34 AS (2 1NFORKATI1ON FOR SEQ ID NO: 41: Mi SEQUE141CF. CHARATEISTICS: LENUGTH: 36 ii., 2

I

*4 a. 4 2 *5 9* an.

a a.

4 5 a.

S

V

.3

TYPE:

STRANDEDNESS:

TOPOLOGY:.

(ii) SEQUENCE DESCRIPTION: SEQ 1D ACACCIJCAGG UACCUUJUAAG ACCAAUGACU

UACAAG

INFORMATION FOR SEQ ID NJO- 42:- SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

a0

STRANDEDNESS:

(El) TOPOLOGY: SEQUENCE DESCRIPTION: SEQ ID GCAGCUGUAG zkucuuAGccA cxmuuuAA GAAAAGGGGG INFORMATION FOR SEQ ID NO: 43: .5 ()SEQUENCE

CHARACTERISTICS:

LENGTK:

TYPE:

STRANDEDNESS:

TOPOLOGY:

o ii SEQUENCE DESCRIPTION; SEQ ID GGGGGACUGG AAGGG INFORMATION FOR TEQ I NO:- 44: SEQUENCE CHARACTERISTICS:

LENGTH:

5 (B3) TYPE:

STRANDEDNESS:

TOPOLOGY:

(i)SEQUENCE DESCRIPTION: SEQ ID NO: -GCUAAUUCAC

UCCCAACGA

o INFORMATION FOR SEQ ,D NO: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STR-ANEDNESS

Ul-SEQUENCE DRSCRIPTION:'SEQD AGAAAGTIAUCc-kUUGAUCU GUGACUAC cCAm nucleic acid single l inear NO: 42.: 36 nucleic acid single' linear NO: 42: nucleic acid single linear NO:- 43: 19 nucleic acid single linear 44: 19 34 nuc-leic acid r5ingle linear NO-- 4S: 34 .1 20 25 INFORM4ATION FOR SEQ ID No; 46: C)SEQUENCE

CHARACTERISTICS:

LENGTH:, 24 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: li-near CiSEQUENCE DESCRIPTION: SEQ ID NO: 46: AUUGGCAGAA CUACAAcA GGAC 24 INFORM ATION FOR SEQ ID No: 47: iiSEQUENJCE

CHARACTERISTICS.

LENGH1: (B3) TYPE: 'nucleic acid STRANDEDNESS: single TopoLOcy, linear (iSEQUENCE DESCRIPTION: SEQ ID NO: 47: 'tCAGAIJAUCC

A

INFORr4ATIOqt FOR SEQ ID NO: 48: SEQUENCE

CHARACTERISTICS*

,LENGTiH: TYPE: nucleic ~i STRAN-DEDNESS: single- TOPOLOGY: linear' (ii) SEQUENCE DESCRIPTION: SEQ ID NO:4.

AAGCUJAGTJTAC CAGUD 1 INFORMATION FOR SEQ ID NO0: 49:i)SEQUENCE

CHARACTERISTICS:

LE-NGTH: 24 TYP2 nucleic acid STRANDEDESS single TOPOLOGY. linear GAi) SEQUENCE DESCRIPTION: SEQ I0 No:49 GCAAACACCA GCUU 149 10-V~IO FOR SEQ Ib No: i)SEQUENCE

CHARACTERISTICS..

LENGTH: 29 TYPE: nucleic acid STRANflEDNR~SS: Single

I

-4 TOPOLOGY., linear (iSEQUENCE DESCRIPTION; SEQ ID No: ACCCUGUGAG CCUGCAUGGAAuGGAUGAC 29.

INFORMATION FOR SEQ ID NO: 51: SEQUENCE CHARACTERISTICS: LENGTH: YPE:nucleic acid STAfENS-single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO:Sl AGZUGGAbGGU UGACAGCCGC XNFO0FMTION FOR SEQ ID NO: 52: i)SEQUENCE

CHARACTERISTICS:

LENGTH- 42 is(1)' TYPE: nucleicacid STRANDEDNESS: sinigle TOPOLOGY; linear (iSEQUENCE DESCRIPTION: SEQ ID NO:. 52: AGUACuuCAA GAACUGCUGAUATJCGAGCrUU GCUACAAGGG AC 42 20 INFORMATION FOR SEQ ID) 1O: 53: SEQUEN~CE CHAACTRITICS-p LENGTH: 17 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY:. linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: .5-4: CUGCU UUUUG CCUGUAC. 17 INFORM~ATION FOR SEQ ID NO): 54; (i.),SEQUENCE

CHARACTERISTICS:

LENGTHd: TYPE-, 1ucleic acid.

STRANDEDNESS: single TOPOLOGY: linear (ii) SEQEC LC~TO SEQ ID No: 54: V 5UCUGAGCCWJ GGAGCUC 1? (2)"INFORIV.T-ON FOR'St ID NO; SEQORCE CHRAACiER

ISV!CS:

/rJ -U-1 C- o: -4 LEN(GTh: 1 TYPE: nucleic acid STRINDEIDN'ESS. single TOPOLOGY: linear .Sj;QUENCE DESCRTLPTION-- SFQ Tr- UA3P'CTiGC

C-

INOMNTION FOR SEQ ID NO:. 56: SEQUENCE CHARACTrERISTICS:

LENGTH:

IB) TYPE;- nuqleic.acid STRANDEDNESS-sple TOPOLOGY: l~a (ii) SEQUENtE DESCRIPTION: SEQ I a: 56:- UGCCUGUAGA vCC-uAGACO- INFORMATION FOR SEQ ID NO' 57: SEQUENCE CHARACTERISVICS: -LENGTH. YPE-nucleic acid S-1YP 11ES: CD) TOPOLOGY, l-.inear (ii) SEQUENCE DESC-RI FTION.: EQ I D NO405, AGCAUCCAGG .AAGUCAGCC 3 INFORMATION FOR SEQ::!2 1Q 58:) SEQUENICE JmTv.'ERI ST ICS:

LEMOTH~

()TYPE: nucleit;-aci.

STRANDEDNESS: inl C)TOPOLOGY: linear Ci QUENCE DESCRIPTION: SQINO 56 40 CAAGUGGUCA J~Gi (2)AINOR14ATION FOR,-ESQ; ID NO: ()SEQUEnLCE CHARACTERISTIrCS- CA) LENGTH: is 4: TYPE: nucleic aci 3S ~C.()RiDflES single (D0.TOPOLOGY; linear (iiD) SEQUENCE D)ESCRIPTION- SEQ ID No- 59.: 14 e 2 a d

A

:6 GGAGCAG:UAU CUCAA INFORMATION FOR SEQ-ID NO: GO: SEQUENCE CHARACTERISTICS: LENGTH: is TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6Q: CCNCAGGUAC CUUUA. INFORMATION FOR SEQ ID NO: 61: SEQUENCE CHARACTERISTICS: LENGTH:. TYPE: nucleic acid STRANDEDNESS:- single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ITD NO! 61: GGGGGACUGG AUGG INFORMAT1ION FOR SEQ ID NO: 62: SEQUENCE CHARACTERISTICS: LENGTH: 1 B)TYPE: nucleic acid Z(C) STRANDEDNBSS: single TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 62: CCAUAUGUAU GUUJC i INFORMATION FOR SEQ ID NO: 63:.

M%(i SEQUENCE CHARACTERISTICS: WA LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO): 63: AGACUGGUAA UAANA 1 INFORMATION FOR SEQ ID NO: 64: Ui) SEQUENCE CHARACTERISTICS: LENGTH:.1 YPE:nucleic acid STRA1NDEDNESS: single TOPOLOGY! linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 64: AUCUGGGUCA GGGAG 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic a STRANDEDNESS: single TOPOLOGY: linear- (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6S: AUUUGGGUtCA GGGAG INFORMATION FOR SEQ ID NO: 66: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic a STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 66: CAGGGAGUCU INFORMATION FOR SEQ ID NO: *67: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic z STRANDEDNESS: single TOPOLOGY: linear *(iiJ) SEQUENCE DESCRIPTION: SEQ ID NO: .67: ACACAAGUAG ACCCU INFORMATION FOR SEQ 1D NO- 68:, SEQUENCE CHARATERISTICS: LENGTH: is TYPE: nucleic STRALMEDNESS: single TOPOLOGY. linear (ii) -sEQUENCE DtSCRIPTIO-SI: SEQ ID NO: 68: AACAAGGUAG GAUCO is ciQ cid .0 'a 44 0 icid acid

Claims (20)

1. 2. An enzymatic nUCleic acid molecule which cleaves of an immunodeficiency virus in a region selected from the group conlsisting of car, rre and the 31 LTR region.
2. 3. The enzymatic nucle *ic acid molecule of claim 1, wherein said RNA molecule is in a hammerhead motif.
3. 4. The enzymatic nucleic acid molecule of claim 1, wherein said RNIA molecule is in a hairpin, hepatitis Delta virus, group i ntron, or R17aseP R1NA motif. An enzymatic nucleic acid molecule which cleaves the sequence shown as any of SEQ. ID. NOS. 1-68, The enzymatic nucleic acid molecul 'e of any of LS claims i-s, wherein said ribozyme comprises between 5 and 23 bas23 complementaryt the RNA of said gene or region.
4. 7. The enzymatic nucleic acid'of claim 6. wherein .said ribozyme -'c-cmprises between 10 and is bases comlemntay t.-,iv.RNA of said gene or region. .08. A mammalian cell icungan enzymaticnuli acid molecule of any of claims The cell of claim wherensi cll sa human c eli. em s c ll i a
5. 10. The cell of claim 9, wherein said celp-is a T4 Slymphocyte having a :CD4 receptor molecule On its cell
6. 11. An expression vector comprising nucleic acid encoding the enzymatic nucleic acid, molecule of any of claims 1-5, in a manner which allows expression of that enzymatic RNA molecule within a mammalian cell.
7. 12. A method for the treatment of an acquired immunodeficiency disease by s administering to a patient an enzymatic nucleic acid molecule of any of claims
8. 13. The method of claim 12, wherein said patient is a human, cat or simian.
9. 14. The ep:tymatic nucleic acid molecule of any one of claims 1-5 when used in the treatment of an acquired immunodeficiency disease. The use of the enzymatic nucleic acid molecule of any one of claim 1-5 in the to preparation of a medicament for use in the treatment of an acquired immunodeficiency disease.
10. 16. A method for providing de,.ctive viral particles comprising the step of contacting a cell infected with an immunc, %fciency virus with an enzymatic nucleic acid molecule active to cleave a gene required for viral replication or infectivity, is 17. The method of claim 16, wherein said molecule is active to cleave a gene required for viral protein synthesis.
11. 18. The method of claim 16, wherein said gene is the nef or rat gene.
12. 19. A defective viral particle produced by the method of any of claims 16, 17 or 18.
13. 20. An imimnogenic preparation comprising a defective viral particle formed by the method of any of claims 16, 17 or 18.
14. 21. A method for immunization against infection by HIV-I, comprising the step of contacting a patient with a vector encoding an enzymatic nucleic acid molecule of any of claims :25 22. The enzymatic nucleic acid molecule of any one of claims 1-5 when used in immunizing a patient against infection by HIV-1. S 23. The use of the enzymatic nucleic acid molecule of any one of claims 1-5 in the preparation of a vaccine for immunizing a patient against infection by HIV-1.
15. 24. Enzymatic nucleic acid molecule which cleaves a substrate sequence shown in o" Table VII or VIII;
16. 25. Enzymatic nucleic acid molecule having a sequence shown in Table VII or VIII.
17. 26. An enzymatic nucleic acid molecule which cleaves RNA of an immunodeficiency virus in nef, substantially as herein before described with reference to any one of the ExaWmles.
18. 27. A method for the treatment of an acquired immunodeficiency disease comprising administring to a patient an enzymatic nucleic acid molecule of claim 26. 28, The enzymatic nucleic acid of claim 26 when used in the treatment of an acquired imnuaodeficiency disease. "Mi'""ti 71
19. 29. The use of the enzymatic nucleic acid mo.Lecule of claim 26 in the preparation of a medicament for use in the treatment of an acquired immunodeicienlcy disease. A method for immunization against 1HV-1 comprising the step of contacting a patient with a vector encoding the enzymatic nucleic acid molecule of claim 26. 3 1. The enzymatic nucleic acid molecule of claim 26 when used in the immunization of a patient against infection by HIV- I.
20. 32. The use of an enzymatic nucleic acid molecule of claim. 26 in the preparation of a vaccine for immunizing a patient against i nfection by HIV- 1. Dated 31 December, 1998 Ribozyme Pharmaceuticals, Inc. Patent Attorneys for the ApplicantlNonifated Person SPRUSON FERGUSON. *4 4* 4* C *4 *4 .q 4. 4 4* S. C C. 0 C 4 4 *4 *4 S. IS C t 'I A jwAit.alot 1*:A!iH'