Method and reagent for inhibiting influenza virus replication

An enzymatic RNA molecule which specifically cleaves an influenza virus RNA.

BACKGROUND OF THE INVENTION
 This invention relates to reagents useful as inhibitors of influenza virus
 replication and infection.
 The following is a discussion of relevant art, none of which is admitted to
 be prior art to the pending claims.
 Three types of influenza viruses (A, B, and C) are distinguishable by
 antigenic reactivities of their internal antigens. There are other
 biological properties which characterize the three types: (a) Influenza A
 viruses have been isolated from many animal species in addition to humans
 while influenza B and C viruses are mainly human pathogens; (b) the
 surface glycoproteins of influenza A exhibit much greater variability than
 their homologues in the B and C viruses; (c) morphological and molecular
 features of C viruses are distinctive from those of the A and B viruses.
 The morphological characteristics of influenza viruses are a genetic trait,
 but spherical morphology dominates after passage in chicken embryos or
 tissue culture. The genes that specify morphology are uncertain, but
 segregate separately from the hemagglutinin (HA) and neuraminidase (NA)
 envelope surface proteins. Within the lipid envelope lies the matrix
 protein (M), which plays a structural function. Within the matrix shell
 are eight single-stranded RNA molecules of negative sense associated with
 the nucleoprotein (NP) and three large proteins (PB1, PB2, and PA)
 required for RNA replication and transcription. At least three viral
 encoded nonstructural proteins (NS1, NS2 and M2) are formed in infected
 cells.
 The organization of the eight RNA segments within the virion has not been
 completely resolved. Although each segment may exist in vivo as a
 nucleoprotein complex, electron microscopic studies have shown that the
 internal component from disrupted virions is a single large helix. The
 virion particle does not seem to be a tight protective coat around the RNA
 because ribonuclease digestion of virion reduces the RNA segments to
 nucleotide. Genomic RNAs of influenza virus are held in a circular
 conformation in a virion and in infected cells by a terminal panhandle
 that plays a role in viral replication. The panhandle structure is present
 in all segments of genomic RNA.
 The HA accounts for 25% of viral protein and is distributed evenly on the
 virion surface. It is responsible for attachment of the virus to cells and
 penetration of virus into cells early in infection. The HA monomer is
 encoded by the fourth largest RNA segment and is synthesized as a single
 polypeptide chain which undergoes posttranslational cleavage at a minimum
 of three sites. Cleavage of the HA polypeptide into HA1 and HA2 is
 necessary for virus particle infectivity. A sequence of 25-32 hydrophobic
 amino acids at the C-terminus of HA2 saves to anchor HA in the virus
 membrane. In spite of functional domain conservation in HA, the amino acid
 or nucleotide sequences of the proteins vary considerably between isolates
 of different subtypes.
 The NA is the second subtype-specific glycoprotein of the virion and is
 composed of a single polypeptide chain. The NA is not evenly distributed
 on the surface of the virion but is found in patches. The role of the NA
 in the life cycle of the virus is unclear. No posttranslational cleavage
 of the NA polypeptide occurs. The nucleotide sequences of different NA
 gene isolates varies considerably between subtypes (e.g., A and B virus
 amino acid homology is 26-29%). The NA gene of influenza B encodes two
 proteins, NA and NB. The NA is thought to be structurally and functionally
 similar to the type A NA. The NB protein is a glycoprotein of unknown
 function which is 100 amino acids in length.
 The nucleoprotein (NP) is one of the type-specific antigens of influenza
 viruses that distinguishes among the influenza type A, B, and C viruses.
 The NP is a multifunctional protein having a structural role in forming
 the nucleoprotein complex and a putative role in transcription and
 replication. Genetic analysis of a large number of influenza strains has
 revealed that the NP genes can be placed into one of five different
 groupings. All avian strains fall within two groups, equine strains fall
 within two more groups and all human and swine strains form the final
 group. The restriction of certain species strains to these groups suggests
 that the NP gene may influence species-specificity or host range.
 RNA segment 7 encodes the two, M proteins (M1 and M2). The mRNA encoding M1
 is colinear with RNA segment 7, whereas M2 is encoded by a spliced mRNA.
 The two proteins share the same initiation codon for protein synthesis and
 the eight amino acid residues before the 5' splice junction of the M2
 mRNA. The remaining 88 amino acids of M2 are encoded in the +1 reading
 frame from nucleotides 740-1104. This organization of RNA segment 7 is
 present in all influenza A and B viruses sequenced.
 The M1 protein is a virion structural protein that is intimately associated
 with the lipid bilayer in close proximity to both glycoproteins and the
 ribonucleoprotein complex. It is also believed to have a role in the
 down-regulation of the virion transcriptase activity. Passively
 transferred monoclonal antibodies to this protein do not confer resistance
 to infection by influenza virus.
 The M2 protein of influenza A is an integral membrane protein that is
 expressed at the surface of infected cells. The M2 protein may be a virion
 associated protein with between 14 and 68 molecules per virion.
 Amantadine-resistant mutants of influenza virus contain mutations in the
 transmembrane domain of the M2 protein. Because amantadine alters viral
 penetration into cells, it is likely that M2 is in the virion.
 Comparison of RNA segment 7 sequences of the H3N2 (Udorn) and H1N1 (PR8)
 strains show that the M protein coding sequences of these viruses
 (isolated 38 years apart) are highly conserved. Lamb, "The genes and
 proteins of influenza viruses," in. Krug. ed. The Influenza Viruses N.Y.,
 Plenum. 1989. Comparison of 230 nucleotides of RNA segment 7 from 5 human
 H1N1, H2N2 and H3N2 strains isolated over a 43 year period suggests that
 the same segment 7 was retained throughout the antigenic shifts of HA and
 NA. Hall and Air, 38 J. Virol. 1, 1981.
 Studies have shown that RNA segment 8 of influenza A and B encodes two
 nonstructural proteins which are translated from separate mRNAs. NS1 and
 NS2 polypeptides of influenza A share 9 amino acids at their N termini,
 after which the NS2 mRNA has a 423 nucleotide deletion; then, the NS2 mRNA
 rejoins the NS1 3' region in the +1 reading frame. NS1 is synthesized in
 large amounts early in infection. NS2 is made only late in infection. Both
 proteins share a nuclear localization signal and can be found in the
 nuclei of infected cells. Large deletions occur in the carboxyl termini of
 the NS1 proteins of field isolates from humans or birds, which indicates
 that a high degree of variation can be tolerated in this polypeptide
 without affecting its function.
 The three largest proteins of the virion (PB1, PB2 and PA) are found
 associated with NP and virion RNA and carry the polymerase activity which
 transcribes invading viral RNA. The PB1 and PB2 proteins form a complex
 when expressed in the absence of other virion proteins or RNA and are
 probably required for complementary RNA synthesis. PA and NP are required
 for virion RNA synthesis. The PB1 gene of influenza B virus shows 61%
 homology with that of the influenza A virus.
 Influenza virus produces an acute febrile infection of the respiratory
 tract characterized by abrupt onset prominent myalgias, headache and
 cough. Pneumonia is the most frequent complication; it may be primary
 viral (due to invasion of lung parenchyma), secondary bacterial, or mixed
 viral and bacterial pneumonia. It may be severe and progressive or mild
 and segmental. Other complications which occur with less frequency include
 Reye's syndrome, myocarditis, pericarditis, myositis, encephalopathy and
 transverse myelitis. It has been estimated that the direct costs of
 influenza exceed $1 billion per year and may reach $3 to $5 billion. Total
 costs may be two to three times higher.
 Two types of vaccines are available for influenza. The "split" vaccines are
 chemically treated to reduce pyrogenic components and are the only type
 given to children under 13 years of age. The "whole" vaccine is generally
 given to adults. Protective antibody titers are present in more than 90%
 of normal subjects after vaccination with influenza A antigens, but there
 is much less response to influenza B antigens. Additionally, elderly
 subjects and patients with renal failures or immunosuppression are at much
 greater risk to infection even with vaccination. The 70-80% efficacy of
 the vaccine is only observed when strain matches are good. Lower efficacy
 is observed when the match is not close, and when patients are
 immunocompromised, or in institutional situations in which virus is
 readily transmitted.
 Two drugs, amantadine and rimantadine, are as effective as influenza
 vaccine in preventing influenza A infections. Unfortunately, they are not
 as active against influenza B, which is responsible for 20% of all
 influenza epidemics and in a given year may be the only virus circulating.
 Amantadine is approved in the United States; rimantadine is not. Both
 drugs appear to impair the uncoating of viral RNA in infected cells by
 blocking the acidification process required to open the viral particles.
 Resistance to amantadine and rimantadine is easily produced in the
 laboratory by serial passage of strains of influenza A virus in low
 concentrations of the drug, and such isolates are cross-resistant to both
 drugs. Drug resistant strains of influenza virus are able to initiate
 infection of cells as effectively as their wild type progenitors.
 Resistance is associated with the presence of point mutations in the RNA
 sequence coding for the M2 protein. This occurs most frequently at amino
 acid 31, but may also occur at positions 27 to 34, which encompass the
 transmembrane domain of the protein. It has been hypothesized that M2
 protein may act as an ion channel to facilitate the acidification of the
 virus particle, and that amantadine and rimantadine block this
 viral-envelope pore.
 SUMMARY OF THE INVENTION
 The invention features novel enzymatic RNA molecules, or ribozymes, and
 methods for their use for inhibiting influenza virus replication. Such
 ribozymes can be used in a method for treatment of diseases caused by
 these related viruses in man and other animals, including other primates.
 Indeed one ribozyme may be designed for treatment of many of the diseases
 caused by these viruses.
 Ribozymes are RNA molecules having an enzymatic activity which is able to
 repeatedly cleave other separate RNA molecules in a nucleotide base
 sequence specific manner. Such enzymatic RNA molecules can be targeted to
 virtually any RNA transcript, and efficient cleavage achieved in vitro.
 Kim et al., 84 Proc. Nat. Acad. of Sci. USA 8788, 1987, Haseloff and
 Gertaek, 334 Nature 585, 1988, Cech, 260 JAMA 3030, 1988, and Jefferies et
 al., 17 Nucleic Acid Research 1371, 1989.
 Ribozymes act by first binding to a target RNA. Such binding occurs through
 the target RNA binding portion of a ribozyme which is held in close
 proximity to an enzymatic portion of the RNA which acts to cleave the
 target RNA. Thus, the ribozyme first recognizes and then binds a target
 RNA through complementary base-pairing, and once bound to the correct
 site, acts enzymatically to cut the target RNA. Strategic cleavage of such
 a target RNA will destroy its ability to direct synthesis of an encoded
 protein. After a ribozyme has bound and cleaved its RNA target it is
 released from that RNA to search for another target and can repeatedly
 bind and cleave new targets.
 The enzymatic nature of a ribozyme is advantageous over other technologies,
 such as antisense technology (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 non-targeted
 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.
 Many regions of the RNAs associated with influenza viruses are appropriate
 targets for ribozyme attack. A ribozyme targeted to these regions in
 influenza viruses may be useful for targeting an equivalent region in
 related viruses whose sequences are unknown.
 There is a high degree of nucleotide sequence homology within the sequences
 of the human and non-human influenza virus genomes and mRNAs. Particularly
 useful regions include sequences in the 5' and 3' regions of the genomic
 RNA segments and complementary regions at the 3' ends of all influenza A
 and B virus mRNAs and their homologous positions in other genomes. In
 addition, the 3' sequence which contributes to the panhandle
 (CCUGCUUUUGCU) is highly conserved among all influenza virus isolates and
 contains two putative ribozyme cleavage sites in a relatively accessible
 region of the RNA. The panhandle sequence (AGUAGAAACAAGG) at the 5' ends
 of the RNAs also contains a suitable ribozyme target site. The
 complementary sequence (CCUUGUUUCUACU) of the 5' panhandle is present at
 the 3' terminus in all mRNAs and contains an additional 4 ribozyme target
 sites which occur within accessible regions of these molecules (i.e., the
 loosely base-paired terminal regions). Detailed information of the
 sequence of the eight RNA segments of influenza (seven in influenza C
 strains) and the molecular weights of the encoded proteins is known.
 Ribozymes targeting any of the above regions of these genomes should be
 able to cleave the RNAs in a manner which will inhibit the translation of
 the molecules.
 Thus, in the first aspect the invention features an enzymatic RNA molecule
 (or ribozyme) which specifically cleaves influenza virus RNA or its
 complementary mRNA.
 Preferred cleavage is at regions required for viral replication (e.g.,
 protein synthesis, such as the regions in RNA segment 7 which regulate or
 encode the M proteins) the conserved 5' and 3' regions of the genomic RNA
 segments which are involved in the panhandle structures, as well as the
 regions at the 3' ends of all mRNAs which are complementary to the 5'
 panhandle structure and their equivalent in other viruses. Alternative
 regions may also be used as targets for ribozyme-mediated cleavage of
 these viral genomes. Each target can be chosen as described below, e.g.,
 by a study of the secondary structure of the RNA, and the individual role
 of such RNA in the replication of the virus. If the targets are contained
 within the open reading frames of regions which encode proteins essential
 to the replication of the virus, then these other targets are preferred
 candidates for cleavage by ribozymes, and subsequent inhibition of viral
 replication. The regions encoding the influenza A virus PB1, PB2 and PA
 proteins and their homologous proteins in the other viruses are examples
 of such preferred targets.
 By "enzymatic RNA molecule" or by "catalytic RNA molecule" it is meant an
 ENA molecule which has complementarity in a substrate binding region to a
 specified gene target, and also has an enzymatic activity which is active
 to specifically cleave RNA in that target. That is, the enzymatic RNA
 molecule is able to intermolecularly cleave RNA and thereby inactivate a
 target RNA molecule. This complementarity functions to allow sufficient
 hybridization of the enzymatic RNA molecule to the target RNA to allow the
 cleavage to occur. 100% complementarity is preferred, but complementarity
 as low as 50-75% may also be useful in this invention. By "equivalent" RNA
 to influenza virus is meant to include those naturally occurring RNA
 molecules associated with viral caused diseases in various animals,
 including humans, and other primates. These viral RNAs have similar
 structures and equivalent genes to each other.
 In preferred embodiments, the enzymatic RNA molecule is formed in a
 hammerhead motif, but may also be formed in the motif of a hairpin,
 hepatitis delta virus, group I intron or RNaseP RNA (in association with
 an RNA guide sequence). Examples of such hammerhead motifs are described
 by Rossi et al., 8 AIDS RESEARCH AND HUMAN RETROVIRUSES 183, 1992, of
 hairpin motifs by Hampel et al., RNA CATALYST FOR CLEAVING SPECIFIC RNA
 SEQUENCES, filed Sep. 20, 1989, which is a continuation-in-part of U.S.
 Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, 28 Biochemistry
 4929, 1989 and Hampel et al., 18 Nucleic Acids Research 299, 1990, and an
 example of the hepatitis delta virus motif is described by Perrotta and
 Been, 31 Biochemistry 16, 1992, of the RNaseP motif by Guerrier-Takada et
 al., 35 Cell 849, 1983, and of the group I intron by Cech et al., U.S.
 Pat. No. 4,987,071. These specific motifs are not limiting in the
 invention and those skilled in the art will recognize that all that is
 important in an enzymatic RNA 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 substrate binding site which impart an RNA cleaving
 activity to the molecule.
 In particularly preferred embodiments, the RNA which is cleaved in
 influenza virus RNA is selected from one or more of the following
 sequences:
 genomic RNAs
 CCUGCUUUUGCU (Seq. ID. No. 1)
 AGUAGAAACAAGG (Seq. ID. No. 2)
 all mRNAs
 CCUUGUUUCUACU (Seq. ID. No. 3)
 M protein mRNAs
 nu-
 cleo-
 tide
 number
 8 GCAGGUAGAUAUUGAAAGATGAG (Seq. ID. No. 4)
 35 CUAACCGAGG (Seq. ID. No. 5)
 64 UAUCGUCCCGUCAGGCC (Seq. ID. No. 6)
 81 CCCUCAAAGCCGAGAUCGCG (Seq. ID. No. 7)
 159 GGCUAAAGACA (Seq. ID. No. 8)
 266 CAAAAUGCCCUAAAUGGGAAUGGAG (Seq. ID. No. 9)
 312 CAGUCAAACUAUACAGGAAACUG (Seq. ID. No. 10)
 331 ACUGAAAAGAGAGAUAA (Seq. ID. No. 11)
 433 AACGGUAACCACA (Seq. ID. No. 12)
 466 GUGUGCCACUUG (Seq. ID. No. 13)
 512 AGACAGAUGGUAACUACUACC (Seq. ID. No. 14)
 537 CACUAAUAAGGCAUGAAAACAG (Seq. ID. No. 15)
 556 CAGAAUGGUGCUG (Seq. ID. No. 16)
 578 ACGGCUAAGGCUAUGGAGCAG (Seq. ID. No. 17)
 626 GAACGCAUGG (Seq. ID. No. 18)
 652 UAGGCAGAUGGUGCAGGCGAUGAGG (Seq. ID. No. 19)
 671 AUGAGGACUAUUGGGACUCACCC (Seq. ID. No. 20)
 691 CCCUAGCUCCAGUG (Seq. ID. No. 21)
 739 GGCCUACCAAAAACGGAUGGGAGUG (Seq. ID. No. 22)
 783 GAUCCUCUCAUUAUUGCC (Seq. ID. No. 23)
 825 UUGAUAUUG (Seq. ID. No. 24)
 840 CUUGAUCGUC (Seq. ID. No. 25)
 863 UAUUUAUCGUCGCCUUAAAUA (Seq. ID. No. 26)
 902 UUCUACGGAAGGAGUGCCU (Seq. ID. No. 27)
 921 GAGUCUAUGAGGGA (Seq. ID. No. 28)
 938 GUAUCGGCAGGAACAACA (Seq. ID. No. 29)
 951 CAACAGAGUGUAGUGG (Seq. ID. No. 30)
 977 UGGUCAUUUU (Seq. ID. No. 31)
 995 AGAGCUGGAGUAAAAACUACCUUG (Seq. ID. No. 32)
 In a second related aspect, the invention features a vertebrate cell which
 includes an enzymatic RNA molecule as described above. Preferably, the
 vertebrate man or other primate cell.
 In a third related aspect, the invention features an expression vector
 which includes nucleic acid encoding the enzymatic RNA molecules described
 above, located in the vector, e.g., in a manner which allows expression of
 that enzymatic RNA molecule within a vertebrate cell.
 In a fourth related aspect, the invention features a method for treatment
 of a influenza virus-caused fisease by administering to a patient an
 enzymatic RNA molecule which cleaves influenza virus RNA, e.g., in the 5'
 panhandle region.
 The invention provides a class of chemical cleaving agents which exhibit a
 high degree of specificity for the viral RNA of influenza virus in
 virus-infected cells or virion particles. The ribozyme molecule is
 preferably targeted to a highly conserved sequence region of an influenza
 virus such that all types and strains of these viruses can be treated with
 a single ribozyme. Such enzymatic RNA molecules can be delivered
 exogenously 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.
 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 utility
 of this approach. In this invention, small ribozyme motifs (e.g., of the
 hammerhead structure, shown generally in FIG. 1) are used for 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 region.
 The enzymatic RNA molecules of this invention can be used to treat
 influenza virus infections. 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 preferred ribozymes disable those
 structures required for successful initiation of viral protein synthesis.
 The preferred targets of the present invention are advantageous over other
 targets since they do not act only at the time of viral absorption or
 genomic replication during infection. 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 virions
 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 influenza viruses to produce viral
 particles which have intact capsids and defective genomic RNA. These
 particles can then be used for instigation of immune responses in a
 prophylactic manner, or as a treatment of infected animals.
 In yet another aspect, the invention features use of influenza viruses as
 vectors for carrying an enzymatic RNA molecule to a cell infected with
 another virus. Such vectors can be formed by standard methodology.
 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 drawing will first briefly be described.
 Drawing
 FIG. 1 is a diagrammatic representation of a hammerhead motif ribozyme
 showing stems I, II and III (marked (I), (II) and (III) respectively)
 interacting with a virus target region. The 5' and 3' ends of both
 ribozyme and target are shown. Dashes indicate base-paired nucleotides.
 Target Sites
 The genome of influenza viruses may be subject to rapid genetic drift by
 virtue of its RNA content and the nature of errors in genomic replication.
 Those regions (genes) of the genome which are essential for virus
 replication, however, are expected to maintain a constant sequence (i.e.,
 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 influenza viruses, and thus only one
 ribozyme is needed to destroy all such viruses. Thus, one ribozyme may be
 used to target all influenza viruses. We have selected several such RNA
 regions of these viruses, and examined their nucleotide sequences for the
 presence of conserved areas which may be cleaved by ribozymes targeted to
 those regions. Three regions analyzed in detail are the 5' and 3'
 panhandle regions, the complementary 3' regions present in all mRNAs, and
 the mRNAs encoding the M proteins; other genes can be analyzed in a manner
 similar to that described below.
 Ribozymes targeting selected regions of the influenza virus genome are
 preferably chosen to cleave the target RNA in a manner which inhibits
 expression of the RNA. Genes are selected such that viral replication is
 inhibited, e.g., by inhibiting protein synthesis or genomic replication
 and packaging into virions. Selection of effective target sites within
 these critical regions of viral RNA entails testing the accessibility of
 the target RNA to hybridization with various oligonucleotide probes. These
 studies can be performed using RNA 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 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
 ribozyme, and then testing each of these sites to determine their
 suitability as targets by ensuring that secondary structure formation is
 minimal.
 The genomic sequences of the viruses are compared in their potential target
 regions. Putative ribozyme cleavage sites are found to be highly conserved
 between strains and species of virus sequence. These sites represent the
 preferred sites for hammerhead or other ribozyme cleavage within these
 target RNAs.
 Short RNA substrates corresponding to each of the gene sites are designed.
 Each substrate is composed of two to three nucleotides at the 5' and 3'
 ends that will not base pair with a corresponding ribozyme recognition
 region. The unpaired regions flank a central region of 12-14 nucleotides
 to which complementary arms in the ribozyme are designed.
 The structure of each substrate sequence is predicted using a PC fold
 computer program. Sequences which give a positive free energy of binding
 are accepted. Sequences which give a negative free energy are modified by
 trimming one or two bases from each of the ends. If the modified sequences
 are still predicted to have a strong secondary structure, they are
 rejected.
 After substrates are chosen, ribozymes are designed to each of the RNA
 substrates. Ribozyme folding is also analyzed using PC fold.
 Ribozyme molecules are sought which form hammerhead motif stem II (see FIG.
 1) regions and contain flanking arms which are devoid of intramolecular
 base pairing. Often the ribozymes are modified by trimming a base from the
 ends of the ribozyme, or by introducing additional base pairs in stem II
 to achieve the desired fold. Ribozymes with incorrect folding are
 rejected. After substrate/ribozyme pairs are found to contain correct
 intramolecular structures, the molecules are folded together to predict
 intermolecular interactions. A schematic representation of a ribozyme with
 its coordinate base pairing to its cognate target sequence is shown in
 FIG. 1.
 Using such analyses, predictions of effective target sites in the viral
 genes, based upon computer generated sequence comparisons, were obtained.
 These are identified as SEQ. ID. NOS. 1-32, shown above.
 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 target
 site without requiring synthesis of a ribozyme. It can be used to screen
 for sites most suited for ribozyme attack.
 Synthesis of Ribozymes
 Ribozymes useful in this invention can be produced by gene transcription as
 described by Cech, supra, or by chemical synthesis. Chemical synthesis of
 RNA is similar to that for DNA synthesis. The additional 2'-OH group in
 RNA, however, requires a different protecting group strategy to deal with
 selective 3'-5' internucleotide bond formation, 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 3'-5' internucleotide linkages, with average coupling yields in
 excess of 99%, and requires only a two-step deprotection of the polymer.
 A method based upon H-phosphonate chemistry gives a relatively lower
 coupling efficiency than a method based upon phosphoramidite chemistry.
 This is a problem for synthesis of DNA as well. A promising approach to
 scale-up of automatic oligonucleotide synthesis has been described
 recently for the H-phosphonates. A combination of a proper coupling time
 and additional capping of "failure" sequences gave high yields in the
 synthesis of oligodeoxynucleotides in scales in the range of 14 micromoles
 with as little as 2 equivalents of a monomer in the coupling step. Another
 alternative approach is to use soluble polymeric supports (e.g.,
 polyethylene glycols), instead of the conventional solid supports. This
 method can yield short oligonucleotides in hundred milligram quantities
 per batch utilizing about 3 equivalents of a monomer in a coupling step.
 Various modifications to ribozyme structure can be made to enhance the
 utility of ribozymes. Such modifications will enhance shelf-life,
 half-life in vitro, stability, and ease of introduction of such ribozymes
 to the target site, e.g., to enhance penetration of cellular membranes,
 and confer the ability to recognize and bind to targeted cells.
 Exogenous delivery of ribozymes benefits from chemical modification of the
 backbone, e.g., by the overall negative charge of the ribozyme molecule
 being reduced to facilitate diffusion across the cell membrane. The
 present strategies for reducing the oligonucleotide charge include:
 modification of internucleotide linkages by ethylphosphonates, use of
 phosphoramidites, linking oligonucleotides to positively charged
 molecules, and creating complex packages composed of oligonucleotides,
 lipids and specific receptors or effectors for targeted cells. Examples of
 such modifications include sulfur-containing ribozymes containing
 phosphorothioates and phosphorodithioates as internucleotide linkages in
 RNA. Synthesis of such sulfur-modified ribozymes is achieved by use of the
 sulfur-transfer reagent, .sup.3 H-1,2-benzenedithiol-3-one 1,1-dioxide.
 Ribozymes may also contain ribose modified ribonucleotides. Pyrimidine
 analogues are prepared from uridine using a procedure employing
 diethylamino sulphur trifluoride (DAST) as a starting reagent. Ribozymes
 can also be either electrostatically or covalently attached to polymeric
 cations for the purpose of reducing charge. The polymer can be attached to
 the ribozyme by simply converting the 3'-end to a ribonucleoside
 dialdehyde which is obtained by a periodate 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 2'-O-methylribose and 5' or 3'
 capping or blocking with m.sub.7 GpppG or m.sub.3.sup.2,2,7 GpppG.
 For example, a kinased ribozyme is contacted with guanosine triphosphate
 and guanyltransferase to add a m.sup.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 liquid chromatography (HPLC). Other liquid
 chromatography techniques, employing reverse phase columns and anion
 exchangers on silica and polymeric supports may also be used.
 There follows an example of the synthesis of one ribozyme. A solid phase
 phosphoramidite chemistry is employed. Monomers used are
 2'-tert-butyl-dimethylsilyl cyanoethylphosphoramidities of uridine,
 N-benzoyl-cytosine, N-phenoxyacetyl adenosine and guanosine (Glen
 Research, Sterling, Va.). Solid phase synthesis is carried out on either
 an ABI 394 or 380B DNA/RNA synthesizer using the standard protocol
 provided with each machine. The only exception is that the coupling step
 is increased from 10 to 12 minutes. The phosphoramidite concentration is
 0.1 M. Synthesis is done on a 1 .mu.mole scale using a 1 .mu.mole RNA
 reaction column (Glen Research). The average coupling efficiencies are
 between 97% and 98% for the 394 model, and between 97% and 99% for the
 380B model, as determined by a calorimetric measurement of the released
 trityl cation.
 Blocked ribozymes are cleaved from the solid support (e.g., CPG), and the
 bases and diphosphoester moiety deprotected in a sterile vial by dry
 ethanolic ammonia (2 mL) at 55.degree. C. for 16 hours. The reaction
 mixture is cooled on dry ice. Later, the cold liquid is transferred into a
 sterile screw cap vial and lyophilized.
 To remove the 2'-tert-butyl-dimethylsilyl groups from the ribozyme, the
 residue is suspended in 1 M tetra-n-butylammonium fluoride in dry THF
 (TBAF), using a 20 fold excess of the reagent for every silyl group, for
 16 hours at ambient temperature (about 15-25.degree. C.). The reaction is
 quenched by adding an equal volume of sterile 1 M triethylamine acetate,
 pH 6.5. The sample is cooled and concentrated on a SpeedVac to half the
 initial volume.
 The ribozymes are purified in two steps by HPLC on a C4 300 .ANG. 5 mm
 DeltaPak column in an acetonitrile gradient.
 The first step, or "trityl on" step, is a separation of 5'-DMT-protected
 ribozyme(s) from failure sequences lacking a 5'-DMT group. Solvents used
 for this step are: A (0.1 M triethylammonium acetate, pH 6.8) and B
 (acetonitrile). The elution profile is: 20% B for 10 minutes, followed by
 a linear gradient of 20% B to 50% B over 50 minutes, 50% B for 10 minutes,
 a linear gradient of 50% B to 100% B over 10 minutes, and a linear
 gradient of 100% B to 0% B over 10 minutes.
 The second step is a purification of a completely deblocked ribozyme by a
 treatment of 2% trifluoroacetic acid on a C4 300 .ANG. 5 mm DeltaPak
 column in an acetonitrile gradient. Solvents used for this second step
 are: A (0.1 M Triethylammonium acetate, pH 6.8) and B (80% acetonitrile,
 0.1 M triethylammonium acetate, pH 6.8). The elution profile is: 5% B for
 5 minutes, a linear gradient of 5% B to 15% B over 60 minutes, 15% B for
 10 minutes, and a linear gradient of 15% B to 0% B over 10 minutes.
 The fraction containing ribozyme is cooled and lyophilized on a SpeedVac.
 Solid residue is dissolved in a minimum amount of ethanol and sodium
 perchlorate in acetone. The ribozyme is 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 (e.g., the human
 cytomegalovirus immediate early promoter) should be chosen to be a
 relatively strong promoter, and expressible both in vitro and in vivo.
 Such a promoter should express the ribozyme at a level suitable to effect
 production of 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 the ribozyme is useful to ensure that the
 required transcription initiation sequence (GG or GGG or GGGAG) does not
 bind to some other part of the ribozyme and thus affect regulation of the
 transcription process. The 5' hairpin is also useful to protect the
 ribozyme from 5'-3' exonucleases. A selected hairpin at the 3' end of the
 ribozyme is useful since it acts as both a transcription termination
 signal, and as a protection from 3'-5' exonucleases. One example of a
 known termination signal is that present on the T7 RNA polymerase system.
 This signal is about 30 nucleotides in length. Other 3' hairpins of
 shorter length can be used to provide good termination and RNA 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 attached.
 Poly(A) tails are also useful to protect the 3' end of the ribozyme. These
 can be provided by either including a poly(A) signal site in the
 expression vector (to signal a cell to add the poly(A) tail in vivo), or
 by introducing a poly(A) sequence directly into the expression vector. In
 the first approach the signal must be located to prevent unwanted
 secondary structure formation with other parts of the ribozyme. In the
 second approach, the poly(A) stretch may reduce in size over time when
 expressed in vivo, and thus the vector may need to be checked over time.
 Care must be taken in addition of a poly(A) tail which binds poly(A)
 binding proteins which prevent the ribozyme from acting upon their target
 sequence.
 Ribozyme Testing
 Once the desired ribozymes are selected, synthesized and purified, they are
 tested in kinetic and other experiments to determine their utility. An
 example of such a procedure is provided below.
 Preparation of Ribozyme
 Crude synthetic ribozyme (typically 350 .mu.g at a time) is purified by
 separation on a 15% denaturing polyacrylamide gel (0.75 mm thick, 40 cm
 long) and visualized by UV shadowing. Once excised, gel slices containing
 full length ribozyme are soaked in 5 ml gel elution buffer (0.5 M NH.sub.4
 OAc, 1 mM EDTA) overnight with shaking at 4.degree. C. The eluent is
 desalted over a C-18 matrix (Sep-Pak cartridges, Millipore, Milford,
 Mass.) and vacuum dried. The dried RNA is resuspended in 50-100 .mu.l TE
 (TRIS 10 mM, EDTA 1 mM, pH 7.2). An aliquot of this solution is diluted
 100 fold into 1 ml TE, half of which is used to spectrophotometrically
 quantitate the ribozyme solution. The concentration of this dilute stock
 is typically 150-800 nM. Purity of the ribozyme is confirmed by the
 presence of a single band on a denaturing polyacrylamide 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 portions are ligated (or otherwise juxtaposed) together. A
 specific example of hammerhead ribozyme synthesis is provided below.
 The method involves synthesis of two (or more) shorter "half" ribozymes and
 ligation of them together using T4 RNA ligase. For example, to make a 34
 mer ribozyme, two 17 mers are synthesized, one is phosphorylated, and both
 are gel purified. These purified 17 mers 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. The 34 mer RNA so formed is then HPLC
 purified.
 Preparation of Substrates
 Approximately 10-30 pmoles of unpurified substrate is radioactively 5'
 end-labelled with T4 polynucleotide kinase using 25 pmoles of
 [.gamma.-.sup.32 P] ATP. The entire labelling mix is separated on a 20%
 denaturing polyacrylamide gel and visualized by autoradiography. The full
 length band is excised and soaked overnight at 4.degree. C. in 100 .mu.l
 of TE (10 mM Tris-HCl pH 7.6, 0.1 mM EDTA).
 Kinetic Reactions
 For reactions using short substrates (between 8 and 16 bases) a substrate
 solution is made 1X in assay buffer (75 mM Tris-HCl.sub.1, pH 7.6; 0.1 mM
 EDTA, 10 MM MgCl.sub.2) such that the concentration of substrate is less
 than 1 nM. A ribozyme solution (typically 20 nM) is made 1X in assay
 buffer and four dilutions are made using 1X assay buffer. Fifteen .mu.l of
 each ribozyme dilution (i.e., 20, 16, 12, 8 and 4 nM) is placed in a
 separate tube. These tubes and the substrate tube are pre-incubated at
 37.degree. C. for at least five minutes.
 The reaction is started by mixing 15 .mu.l of substrate into each ribozyme
 tube by rapid pipetting (note that final ribozyme concentrations are 10,
 8, 6, 4, 2 nM). 5 .mu.l aliquots are removed at 15 or 30 second intervals
 and quenched with 5 .mu.l stop solution (95% formamide, 20 mM EDTA xylene
 cyanol, and bromphenol blue dyes). Following the final ribozyme time
 point, an aliquot of the remaining substrate is rmoved as a zero ribozyme
 control.
 The samples are separated on either 15% or 20% polyacrylamide gels. Each
 gel is visualized and quantitated with an Ambis beta scanner (Ambis
 Systems, San Diego, Calif.).
 For the most active ribozymes, kinetic analyses are performed in substrate
 excess to determine K.sub.m and K.sub.cat values.
 For kinetic reactions with long RNA substrates (greater than 15 bases in
 length) the substrates are prepared by transcription using T7 RNA
 polymerase and defined templates containing a T7 promoter, and DNA
 encoding appropriate nucleotides of the viral RNA. The substrate solution
 is made lx in assay buffer (75 mM Tris-HCl, pH 7.6; 0.1 mM EDTA; 10 mM
 MgCl.sub.2) and contains 58 nanomolar concentration of the long RNA
 molecules. The reaction is started by addition of gel purified ribozymes
 to 1 .mu.M concentration. Aliquots are removed at 20, 40, 60, 80 and 100
 minutes, then quenched by the addition of 5 .mu.l stop solution. Cleavage
 products are separated using denaturing PAGE. The bands are visualized and
 quantitated with an Ambis beta scanner.
 Kinetic Analysis
 A simple reaction mechanism for ribozyme-mediated cleavage is:
 ##EQU1##
 where R=ribozyme, S=substrate, and P=products. The boxed step is important
 only in substrate excess. Because ribozyme concentration is in excess over
 substrate concentration, the concentration of the ribozyme-substrate
 complex ([R:S]) is constant over time except during the very brief time
 when the complex is being initially formed, i.e.,:
 ##EQU2##
 where t=time, and thus:
EQU (R) (S)k.sub.1 =(RS) (k.sub.2 +k.sub.1).
 The rate of the reaction is the rate of disappearance of substrate with
 time:
 ##EQU3##
 Substituting these expressions:
 ##EQU4##
 Integrating this expression with.respect to time yields:
 ##EQU5##
 where S.sub.0 =initial substrate. Therefore, a plot of the negative log of
 fraction substrate uncut versus time (in minutes) yields a straight line
 with slope:
 ##EQU6##
 where k.sub.obs =observed rate constant. A plot of slope (k.sub.obs) versus
 ribozyme concentration yields a straight line with a slope which is:
 ##EQU7##
 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. An example of preparing ribozymes in a liposome
 delivery vehicle is given below.
 Liposome Preparation
 Lipid molecules are dissolved in a volatile organic solvent (CHCl.sub.3,
 methanol, diethylether, ethanol, etc.). The organic solvent is removed by
 evaporation. The lipid is hydrated into suspension with 0.lx phosphate
 buffered saline (PBS), then freeze-thawed 3x using liquid nitrogen and
 incubation at room temperature. The suspension is extruded sequentially
 through a 0.4 .mu.m, 0.2 .mu.m and 0.1 .mu.m polycarbonate filters at
 maximum pressure of 800 psi. The ribozyme is mixed with the extruded
 liposome suspension and lyophilized to dryness. The lipid/ribozyme powder
 is rehydrated with water to one-tenth the original volume. The suspension
 is diluted to the minimum volume required for extrusion (0.4 ml for 1.5 ml
 barrel and 1.5 ml for 10 ml barrel) with 1xPBS and re-extruded through 0.4
 .mu.m, 0.2 .mu.m, 0.1 .mu.m polycarbonate filters. The liposome entrapped
 ribozyme is separated from untrapped ribozyme by gel filtration
 chromatography (SEPHAROSE CL-4B, BIOGEL A5M). The liposome extractions are
 pooled and sterilized by filtration through a 0.2 .mu.m filter. The free
 ribozyme was pooled and recovered by ethanol precipitation. The liposome
 concentration is determined by incorporation of a radioactive lipid. The
 ribozyme concentration is determined by labeling with .sup.32 P. Rossi et
 al., 1992 supra (and references cited therein) describe other methods
 suitable for preparation of liposomes.
 In Vivo Assay
 The efficacy of action of a chosen ribozyme may be tested in vivo by use of
 cell cultures sensitive to a selected influenza virus, using standard
 procedures. For example, monolayer cultures of virus-sensitive cells are
 grown in 6 or 96 well tissue culture plates. Prior to infection with
 influenza virus, cultures are treated for 3 to 24 hours with
 ribozyme-containing liposomes. Cells are then rinsed with phosphate
 buffered saline (PBS) and virus added at a multiplicity 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 and medium changes. Virus is harvested from cells into the
 overlying medium. Cells are broken by three cycles of incubation at
 -70.degree. C. and 37.degree. C. for 30 minutes at each temperature, and
 viral titers determined by plaque assay using established procedures.
 These procedures can be modified for each specific virus to be tested.
 Ribonuclease Protection Assay
 The accumulation of target mRNA in cells or the cleavage of the RNA by
 ribozymes or RNaseH (in vitro or in vivo) can be quantified using an RNase
 protection assay.
 In this method, antisense riboprobes are transcribed from template DNA
 using T7 RNA polymerase (U.S. Biochemicals) in 20 .mu.l reactions
 containing 1X transcription buffer (supplied by the manufacturer), 0.2 mM
 ATP, GTP and UTP, 1 U/.mu.l pancreatic RNase inhibitor (Boehringer
 Mannheim Biochemicals) and 200 .mu.Ci .sup.32 P-labeled CTP (800 Ci/mmol,
 New England Nuclear) for 1 h at 37.degree. C. Template DNA is digested
 with 1 U RNase-free DNase I (U.S. Biochemicals, Cleveland, Ohio) at
 37.degree. C. for 15 minutes 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.degree.
 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 microcentrifuge, and lysed in 70 .mu.l of hybridization buffer
 (4 M guanidine isothiocyanate, 0.1% sarcosyl, 25 mM sodium citrate, pH
 7.5). Cell lysate (45 .mu.l) or defined amounts of in vitro transcript
 (also in hybridization buffer) is then combined with 5 .mu.l of
 hybridization buffer containing 5.times.10.sup.5 cpm of each antisense
 riboprobe in 0.5 ml EPPENDORF tubes, overlaid with 25 .mu.l mineral oil,
 and hybridization accomplished by heating overnight at 55.degree. C. The
 hybridization reactions are diluted into 0.5 ml RNase solution (20 U/ml
 RNase A, 2 U/ml RNase T1, 10 U/ml RNase-free DNAse I in 0.4 M NaCl),
 heated for 30 minutes at 37.degree. C., and 10 .mu.l of 20% SDS and 10
 .mu.l of Proteinase K (10 mg/ml) added, followed by an additional 30
 minutes incubation at 37.degree. C. Hybrids are partially purified by
 extraction with 0.5 ml of a 1:1 mixture of phenol/chloroform; aqueous
 phases are combined with 0.5 ml isopropanol, and RNase-resistant hybrids
 pelleted for 10 minutes at room temperature (about 20.degree. C.) in a
 microcentrifuge. Pellets are dissolved in 10 .mu.l loading buffer (95%
 formamide, 1X TBE, 0.1% bromophenol blue, 0.1% xylene cylanol), heated to
 95.degree. C. for five minutes, cooled on ice, and analyzed on 4%
 polyacrylamide/7 M urea gels under denaturing conditions.
 Ribozyme 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
 various chemical modifications (e.g., 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 5' (kinased) and/or
 3' labeled ribozyme, 15 .mu.g of cytosolic extract and 2.5 mM MgCl.sub.2
 in a total volume of 100 .mu.l. The reaction is incubated at 37.degree. C.
 Eight .mu.l aliquots are taken at timed intervals and mixed with 8 .mu.l
 of a stop mix (20 mM EDTA, 95% formamide). Samples are separated on a 15%
 acrylamide sequencing gel, exposed to film, and scanned with an Ambis.
 A 3'-labelled ribozyme can be formed by incorporation of the .sup.32
 P-labeled cordycepin at the 3' OH using poly(A) polymerase. For example,
 the poly(A) polymerase reaction contains 40 mM Tris, pH 8, 10 mM
 MgCl.sub.2, 250 mM NaCl, 2.5 mM MnCl.sub.2 ; 3 .mu.l P.sup.32 cordycepin,
 500 Ci/mM; and 6 units poly(A) polymerase in a total volume of 50 .mu.l.
 The reaction mixture is incubated for 30 minutes at 37.degree. C.
 Effect of Base Substitution Upon Ribozyme Activity
 To determine which primary structural characteristics could change ribozyme
 cleavage of substrate, minor base changes can be made in the substrate
 cleavage region recognized 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 motif. The K.sub.cat /K.sub.m values
 for cleavage using each substrate are then analyzed to determine if such a
 change increases ribozyme cleavage rates. Similar experiments can be
 performed to address the effects of changing bases complementary to the
 ribozyme binding arms. Changes predicted to maintain strong binding to the
 complementary substrate are preferred. Minor changes in nucleotide content
 can alter ribozyme/substrate interactions in ways which are unpredictable
 based upon binding strength alone. Structures in the catalytic core region
 of the ribozyme recognize trivial changes in either substrate structure or
 the three dimensional structure of the ribozyme/substrate complex.
 To begin optimizing ribozyme design, the cleavage rates of ribozymes
 containing varied arm lengths, but targeted to the same length of short
 RNA substrate can be tested. Minimal arm lengths are required and
 effective cleavage varies with ribozyme/substrate combinations.
 The cleavage activity of selected ribozymes can be assessed using
 picornavirus RNA substrates. The assays are performed in ribozyme excess
 and approximate K.sub.cat /K.sub.min values obtained. Comparison of values
 obtained with short and long substrates indicates utility in vivo of a
 ribozyme.
 Intracellular Stability of Liposome-delivered Ribozymes
 To test the stability of a chosen ribozyme in vivo the following test is
 useful. Ribozymes are .sup.32 P-end labeled, entrapped in liposomes and
 delivered to influenza virus sensitive cells for three hours. The cells
 are fractionated and purified by phenol/chloroform extraction. Cells
 (1.times.10.sup.7, T-175 flask) are scraped from the surface of the flask
 and washed twice with cold PBS. The cells are homogenized by douncing 35
 times in 4 ml of TSE (10 mM Tris, pH 7.4, 0.25 M Sucrose, mM EDTA). Nuclei
 are pelleted at 100xg for 10 minutes. Subcellular organelles (the membrane
 fraction) are pelleted at 200,000xg for two hours using an SW60 rotor. The
 pellet is resuspended in 1 ml of H buffer (0.25 M Sucrose, 50 mM HEPES, pH
 7.4). The supernatant contains the cytoplasmic fraction (in approximately
 3.7 ml). The nuclear pellet is resuspended in 1 ml of 65% sucrose in TM
 (50 mM Tris, pH 74., 2.5 mM MgCl.sub.2) and banded on a sucrose step
 gradient (1 ml nuclei in 65% sucrose TM, 1 ml 60% sucrose TM, 1 ml 55%
 sucrose TM, 50% sucrose TM, 300 ul 25% sucrose TM) for one hour at
 37,000xg with an SW60 rotor. The nuclear band is harvested and diluted to
 10% sucrose with TM buffer. Nuclei are pelleted at 37,000xg using an SW60
 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 modifications 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 activity known to
 exist in that fraction.
 The various cell fractions are frozen at -70.degree. C. and used to
 determine relative nuclease resistances of modified ribozyme molecules.
 Ribozyme molecules may be synthesized with 5 phosphorothioate (ps), or
 2'-O-methyl (2'-OMe) modifications at each end of the molecule. These
 molecules and a phosphodiester version of the ribozyme are end-labeled
 with .sup.32 P and ATP using T4 polynucleotide kinase. Equal
 concentrations are added to the cell cytoplasmic 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 .beta.-scanner. The results show whether
 the ribozymes are digested by the cytoplasmic extract, and which versions
 are relatively more nuclease resistant. Modified ribozymes 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 ribozymes can be used in the
 assays. These experiments can also be performed with human cell extracts
 to verify the observations.
 Administration of Ribozyme
 Selected ribozymes can be administered prophylactically, or to virus
 infected patients, e.g., by exogenous delivery of the ribozyme to an
 infected tissue by means of an appropriate delivery vehicle, e.g., a
 liposome, a controlled release vehicle, by use of iontophoresis,
 electroporation or ion paired molecules, or covalently attached adducts,
 and other pharmacologically approved methods of delivery. Routes of
 administration include intramuscular, aerosol, oral (tablet or pill form),
 topical, systemic, ocular, intraperitoneal and/or intrathecal. 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 naked ribozyme uptake with regard to intracellular
 localization, followed by demonstration of efficacy. Alternatively,
 delivery to these same cells in an organ or tissue of an animal can be
 pursued. Uptake studies will include uptake assays to evaluate cellular
 oligonucleotide uptake, regardless of the delivery vehicle or strategy.
 Such assays will also determine the intracellular localization of the
 ribozyme following uptake, ultimately establishing the requirements for
 maintenance of steady-state concentrations within the cellular compartment
 containing the target sequence (nucleus and/or cytoplasm). Efficacy and
 cytotoxicity can then be tested. Toxicity will not only include cell
 viability but also cell function.
 Some methods of delivery that may be used include:
 a. encapsulation in liposomes,
 b. transduction by retroviral vectors,
 c. conjugation with cholesterol,
 d. localization to nuclear compartment utilizing antigen binding site found
 on most snRNAs,
 e. neutralization of charge of ribozyme by using nucleotide derivatives,
 and
 f. use of blood stem cells to distribute ribozymes throughout the body.
 At least three types of delivery strategies are useful in the present
 invention, including: ribozyme 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 be modified
 essentially at random, in ways which reduces its charge but maintains
 specific functional groups. This results in a molecule which is able to
 diffuse across the cell membrane, thus removing the permeability barrier.
 Modification of ribozymes to reduce charge is just one approach to enhance
 the cellular uptake of these larger molecules. The random approach,
 however, is not advisable since ribozymes are structurally and
 functionally more complex than 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 enhance cellular delivery.
 The modifications are also designed to reduce susceptibility to nuclease
 degradation. Both of these characteristics should greatly improve the
 efficacy of the ribozyme. Cellular uptake can be increased by several
 orders of magnitude without 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 between DNA and RNA make this a
 feasible approach. In the body, maintenance of an external concentration
 will be necessary to drive the diffusion of the modified 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 infection, can protect the ribozyme from
 degradative processes.
 Drug delivery vehicles are effective for both systemic and topical
 administration. They can be designed to serve as a slow release reservoir,
 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
 vehicles which fall into this category are liposomes, hydrogels,
 cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
 From this category of delivery systems, liposomes are preferred. Liposomes
 increase intracellular stability, increase uptake efficiency and improve
 biological activity.
 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 internal 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.
 Liposomes offer several advantages: They are non-toxic and biodegradable in
 composition; they display long circulation half-lives; and recognition
 molecules can be readily attached to their surface for targeting to
 tissues. Finally, cost effective manufacture of liposome-based
 pharmaceuticals, either in a liquid suspension or lyophilized product, has
 demonstrated the viability of this technology as an acceptable drug
 delivery system.
 Other 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
 pharmaceuticals, and consequently, can be adapted for ribozyme delivery.
 Topical administration of ribozymes is advantageous since it allows
 localized concentration at the site of administration with minimal
 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 administration routes. Effective
 delivery requires the ribozyme 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 modified ribozyme can be co-formulated
 with permeability enhancers, such as Azone or oleic acid, in a liposome.
 The liposomes can either represent a slow release presentation vehicle in
 which the modified ribozyme and permeability enhancer transfer from the
 liposome into the infected cell, or the liposome phospholipids can
 participate directly with the modified ribozyme and permeability enhancer
 in facilitating cellular delivery. In some cases, both the ribozyme and
 permeability enhancer can be formulated into a suppository formulation for
 slow release.
 Ribozymes may also be systemically administered. Systemic absorption refers
 to the accumulation of drugs in the blood stream followed by distribution
 throughout the entire body. Administration routes which lead to systemic
 absorption include: intravenous, subcutaneous, intraperitoneal,
 intranasal, intrathecal and ophthalmic. Each of these administration
 routes expose the ribozyme 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 into the
 circulation has been shown to be a function of molecular weight or size.
 The use of a liposome or other drug carrier localizes the ribozyme 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.
 A liposome formulation which can deliver ribozymes to lymphocytes and
 macrophages is also useful for the initial site of influenza virus
 replication is in tissues of the nasopharynx and respiratory system.
 Coating of lymphocytes with liposomes containing ribozymes will target the
 ribozymes to infected cells expressing viral surface antigens. Whole blood
 studies show that the formulation is taken up by 90% of the lymphocytes
 after 8 hours at 37.degree. C. Preliminary biodistribution and
 pharmacokinetic studies yielded 70% of the injected dose/gm of tissue in
 the spleen after one hour following intravenous administration.
 Intraperitoneal administration also leads to entry into the circulation,
 with once again, the molecular weight or size controlling the rate of
 entry.
 Liposomes injected intravenously show accumulation in the liver, lung and
 spleen. The composition and size can be adjusted so that this accumulation
 represents 30% to 40% of the injected dose. The rest is left to circulate
 in the blood stream for up to 24 hours.
 The chosen method of delivery should result in cytoplasmic accumulation and
 molecules should have some nuclease-resistance for optimal dosing. Nuclear
 delivery may be used but is less preferable. Most preferred delivery
 methods include liposomes (10-400 nm), hydrogels, controlled-release
 polymers, microinjection or electroporation (for ex vivo treatments) and
 other pharmaceutically applicable vehicles. The dosage will depend upon
 the disease indication and the route of administration but should be
 between 100-200 mg/kg of body weight/day. The duration of treatment will
 extend through the course 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 prolong the intracellular half-life of the ribozyme.
 Thus, chemically modified ribozymes, e.g., with modification of the
 phosphate backbone, or capping of the 5' and 3' ends of the ribozyme with
 nucleotide analogs may require different dosaging. Descriptions of useful
 systems are provided in the art cited above, all of which is hereby
 incorporated by reference herein.
 The claimed ribozymes are also useful as diagnostic tools to specifically
 or non-specifically detect the presence of a target RNA in a sample. That
 is, the target RNA, if present in the sample, will be specifically cleaved
 by the ribozyme, and thus can be readily and specifically detected as
 smaller RNA species. The presence of such smaller RNA species is
 indicative of the presence of the target RNA in the sample.
 Other embodiments are within the following claims.