WO1995004818A1 - Method and reagent for inhibiting human immunodeficiency virus replication - Google Patents
Method and reagent for inhibiting human immunodeficiency virus replication Download PDFInfo
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- WO1995004818A1 WO1995004818A1 PCT/US1994/008613 US9408613W WO9504818A1 WO 1995004818 A1 WO1995004818 A1 WO 1995004818A1 US 9408613 W US9408613 W US 9408613W WO 9504818 A1 WO9504818 A1 WO 9504818A1
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Definitions
- AIDS Acquired immunodeficiency syndrome
- AZT drug azidothymidine
- DDC dideoxycytidine
- RNA in addition to being an informa- tional molecule, can also possess enzymatic activity.
- a rational approach to the problem of target selection involves the following criteria. First, one should select a functionally important target, such as tat, rev, int, psi (packaging site) , or the tRNA lys priming site. Once a gene or target region has been decided upon, the nucleotide sequence should be assessed for strong conservation of sequence among the various isolates. Within these conserved regions, the potential cleavage sites, prefer- ably GUC or GUA (others will suffice, but appear to be less efficiently cleaved) should be chosen. The region should be examined for potential secondary structures, and then the most promising sites chosen.
- ribozymes designed to cleave the integrase gene of HIV. They state that when the ribozyme is tran ⁇ scribed from a plasmid in E. coli it leads to destruction of the integrase RNA and complete blockage of integrase protein synthesis. They state that the HIV-1 integrase gene may be a useful target for therapeutic ribozymes.
- ribozymes in the hammerhead family targeted to HIV-1 gag transcripts. They state that cells challenged with HIV-1 showed a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, and that the reduction in gag RNA was reflected by a reduction in antigen p24 levels. They state that the results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.
- RNA Sequences filed September 20, 1989, which is a continuation-in-part of U.S. Serial No. 07/247,100, filed September 20, 1988, describe hairpin ribozymes, and provides an example of such a ribozyme apparently specific to the gag gene of HIV-1.
- Hampel and Tritz, 28 Biochemistry 4929, 1989 and Hampel et al . , 18 Nucleic Acids Research 299, 1990 also describe hairpin catalytic RNA models and state that one target site is the tat gene in HIV-1.
- Goldberg et al. WO 91/04319 and Robertson and Goldberg WO 91/04324, describe ribozymes expressed within a hepatitis delta vector and state that the genome of the delta virus may carry a ribozyme against the env or gag mRNA of HIV.
- Rossi et al . , WO 91/03162 describe chimeric DNA-RNA catalytic sequences used to cleave HIV-1 gag- transcript or the 5' LTR splice site.
- 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 has been achieved in vitro. Kim et al . , 84 Proc. Natl. Acad.
- 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 enzy ⁇ matic 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.
- ribozyme 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 thera ⁇ Commissionic treatment is lower than that of an antisense oligonucleotide.
- This advantage reflects the ability of the ribozyme to act enzymatically.
- a single ribo ⁇ zyme molecule is able to cleave many molecules of target RNA.
- 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 cleav- age 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.
- the invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting immunodeficiency virus replication, e.g. , HIV-1, HIV-2 and related viruses including FIV-1 and SIV-1.
- 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.
- the ribozyme molecules described are targeted to the LTR, nef, vif, tat and rev viral genes or regions . These genes are known in the art, see, e.g. , Matsukura et al . , 86 Proc. Natl. Acad.
- the invention features an enzymatic RNA molecule (or ribozyme) which cleaves HIV-1 RNA, or its equivalent, regions required for viral repli ⁇ cation, e.g. , protein synthesis, e.g. , the vif, nef, tat or rev gene regions, or at structures known to regulate viral gene expression, e.g., tar, rre or 3' -LTR regions.
- enzymatic RNA molecule it is meant an RNA 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.
- the enzymatic RNA molecule is able to intermolecularly cleave RNA and thereby inact- ivate a target RNA molecule.
- This complementarity func ⁇ tions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur.
- One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.
- “equivalent" RNA to HIV-1 is meant to include those naturally occurring RNA molecules associated with immunodeficiency diseases in various animals, includ ⁇ ing humans, felines, and simians. These viral RNAs have similar structures and equivalent genes to each other, including the vif, nef, tat and rev genes.
- 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-like RNA (in association with an RNA guide sequence) . Examples of such hammerhead motifs are described by Rossi et al. (see citations above) , of hairpin motifs by Hampel et al .
- RNA molecules of this invention 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.
- the RNA 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 sequence folded began at nucleotide number one of the 2.3 kb subgenomic mRNA. This region includes the coding regions for the vif, vpr, vpu, tat, rev, and nef gene products.
- the invention features a mammalian cell which includes an enzymatic RNA molecule as described above.
- the mammalian cell is a human cell, for example, a T4 lymphocyte having a CD4 receptor molecule on its cell surface.
- 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 mammalian cell.
- the invention features a method for treatment of human immunodeficiency disease by administering to a patient an enzymatic RNA molecule which cleaves HIV-1 RNA or related RNA in the vif, nef, tat or rev gene regions.
- the invention features treatment of cats or simians with ribozymes of this invention.
- ribozymes may be identical to those able to cleave HIV-1 RNA, or may be modified to target analogous locations in FIV and SIV virus RNAs .
- 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.
- 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 vi tro transcript having a length of 142 nucleo ⁇ tides. Synthesis of ribozymes greater than 100 nucleo ⁇ tides in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
- ribozymes by expression vectors are primarily feasible using only ex vivo treatments. This limits the utility of this approach.
- an alterna ⁇ tive approach uses smaller ribozyme motifs (e.g. , of the hammerhead structure, shown generally in Fig. 1) and exo ⁇ genous delivery.
- the simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure.
- 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 also be extended to other related viruses 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 transcrip ⁇ tion during infection, but also in latently infected cells and in virally transformed cells.
- 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.
- 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.
- the enzymatic RNA molecules of this invention can be used in vi tro in a cell culture infected with HIV-1 viruses, or related 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.
- 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, e.g. , with DNA or vectors encoding a ribozyme of this invention under the control of a suitable promoter. Diagnostic 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 three- dimensional 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 vi tro, as well as in cells and tissues . Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role
- ribozymes of this invention include detection 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.
- 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.
- synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme effi ⁇ ciencies in the reactions and the absence of cleavage of 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 R ⁇ As in the sample population.
- 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 R ⁇ Ase protection assay so that full-length and cleavage fragments of each R ⁇ A 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 R ⁇ As and putative risk of the desired phenotypic changes in target cells.
- the expression of mR ⁇ A whose protein product is implicated in the development of the phenotype (i.e., HIV) is adequate to establish risk.
- Fig. 1 is a diagrammatic representation of a hammer ⁇ head motif ribozyme showing stems I, II and III (marked (I) , (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.
- Figs. 3A - 3C are diagrammatic representations of three ribozymes of this invention.
- Figs. 4A - 4G are diagrammatic representations of chemically modified ribozymes of this invention (solid circles indicate modified bases) .
- Fig. 4A is unmodified HCH-r37
- Fig. 4B is Thio-substituted HCH- r37S2
- Fig. 4C is Thio-substituted 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 (circled bases are modified with 2' -O-methyl) , specifically the ribozyme is 2' -0-methyl HCH-037A.
- Figs. 6a-6f are graphical representations of ribozyme 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 nuclease resistance.
- Fig. 10 is a graphical representation of activity of various LTR-targeted ribozymes.
- Fig. 12 is a 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 hammer ⁇ head 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.
- HIV-1 The genome of HIV-1 is subject to rapid genetic drift by virtue of its RNA content and the nature of errors in reverse transcription. Those regions (genes) of the genome which are essential for virus replication, however,
- RECTIFIED SHEET (RULE 91) 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 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.
- 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, e.g. , by inhibiting protein synthesis. Selection of effective target sites within these critical regions of HIV-1 RNA entails testing the accessibility of the target RNA to hybridization with various oligonucleo ⁇ tide probes. These studies can be performed using RNA probes and assaying accessibility by cleaving the hybrid molecule with RNaseH (see below) .
- RNAs RNAs
- PAGE polyacrylamide gel electrophoresis
- 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 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 RNAs.
- nef gene sites 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 HIV-1 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 RNAs. In a similar manner, a number of the vif target sites are present in the pol, tat and vpr mRNAs (see Fig. 2) .
- 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 central region of 12-14 nucleotides to which complementary arms in the ribozyme were designed.
- each substrate sequence was predicted using a standard commercially available PC fold 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, they were rejected.
- ribozymes were designed to each of the RNA substrates. Ribozyme folding was also analyzed using PC fold.
- Ribozyme molecules were sought which formed hammer- head motif stem II (see Fig. 1) regions and contained flanking arms which were devoid of intramolecular base pairing. Often the ribozymes were 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 were rejected. After substrate/ribozyme pairs were found to contain correct intramolecular structures, the molecules were 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.
- ribonuclease H 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.
- 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 reproduc- ibly 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 exhibits 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 ⁇ moles with as little as 2 equivalents of a monomer in the coup ⁇ ling 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.
- ribozyme structure can be made to enhance the utility of ribozymes.
- Such modifica ⁇ tions will enhance shelf-life, half-life in vi tro, 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 chemi ⁇ cal 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 methylphosphonates, 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.
- Ribozymes may also contain ribose modified ribonucleotides.
- Pyrimidine analogues are prepared from uridine using a procedure employing diethyl- amino sulphur trifluoride (DAST) as a starting reagent.
- Ribozymes can also be either electrostatically or cova ⁇ lently 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 ribonucleo- side dialdehyde which is obtained by a periodate cleavage of the terminal 2',3'-cis diol system.
- other possible modifications may include different linker arms containing carboxyl, amino or thiol functionalities.
- Yet further examples include use of methylphosphonates and 2'-0- methylribose and 5' or 3' capping or blocking with m 7 GpppG or m 3 2,2,7 GpppG.
- a kinased ribozyme is contacted with guanosine triphosphate and guanyltransferase to add a m 3 G cap to the ribozyme.
- 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 proce ⁇ dures 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) .
- HPLC high pressure liquid chromatography
- Other liquid chromatography tech ⁇ niques, employing reverse phase columns and anion exchangers on silica and polymeric supports may also be used.
- ABI 394 or 380B DNA/RNA 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 ⁇ mole scale using a 1 ⁇ mole RNA reaction column (Glen Research) . The average coupling efficiencies were 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.
- the blocked ribozymes were cleaved from the solid support (e.g. , CPG) , and the bases and diphosphoester moiety deprotected in a sterile vial by incubation in dry ethanolic ammonia (2 mL) at 55°C for 16 hours. The reaction mixture was cooled on dry ice. Later, the cold liquid was transferred into a sterile screw cap vial and lyophilized.
- the solid support e.g. , CPG
- dry ethanolic ammonia 2 mL
- the obtained residue was 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°C) .
- THF dry THF
- the ribozymes were purified in two steps by HPLC on a C4 300 A 5 ⁇ m DeltaPak column in an acetonitrile gradient.
- the first step was a separation 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) and B (aceto ⁇ nitrile) .
- the elution profile was: 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 was a purification of a completely deblocked ribozyme by a treatment of 2% trifluoroacetic acid on a C4 300 A 5 ⁇ m DeltaPak column in an acetonitrile gradient.
- Solvents used for this second step were: A (0.1 M Triethylammonium acetate, pH 6.8) and B (80% aceto ⁇ nitrile, 0.1 M triethylammonium acetate, pH 6.8) .
- the elution profile was: 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 .
- ribozymes 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 region (HCMV iel) promoter
- HCMV iel human cytomegalovirus immediate early region
- 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 transcrip ⁇ tion 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 gene is useful since it acts as a transcription termination signal, and as a protection from 3' -5' exonuclease activity.
- a known termination signal is that present on the T7 RNA polymer- ase 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 includ ⁇ ing 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 expres ⁇ sion 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.
- ribozymes Once the desired ribozymes are selected, synthesized and purified, they are tested in kinetic and other experi ⁇ ments to determine their utility. An example of such a procedure is provided below.
- Crude synthetic ribozyme (typically 350 ⁇ g at a time) is purified by separation on a 15% denaturing polyacryl ⁇ amide 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 4 0Ac, 1 mM EDTA) overnight with shaking at 4°C. The eluent is desalted over a C-18 matrix (Sep-Pak cartridges, Millipore, Milford, MA) and vacuum dried.
- 5 ml gel elution buffer 0.5 M NH 4 0Ac, 1 mM EDTA
- RNA is resuspended in 50-100 ⁇ 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 spectrophotometric- ally 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 gener ⁇ ally 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 juxta ⁇ posed) 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.
- T4 RNA ligase For example, to make a 34 mer ribo ⁇ zyme, 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. Alternatively, the DNA splint strand may be omitted from the ligation reaction if the complementary binding affects favorable ligation of the two RNA molecules. The 34 mer RNA so formed is then HPLC purified.
- a substrate solution is made IX in assay buffer (75 mM Tris-HCl, pH 7.6; 0.1 mM EDTA, 10 mM MgCl 2 ) 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 ⁇ 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°C for at least five minutes.
- the reaction is started by mixing 15 ⁇ l of substrate into each ribozyme tube by rapid pipetting (note that final ribozyme concentrations are 10, 8, 6, 4, 2 nM) .
- Five ⁇ l aliquots are removed at 15 or 30 second intervals and quenched with 5 ⁇ l stop solution (95% formamide, 20 mM EDTA xylene cyanol, and bromphenol blue dyes) .
- 5 ⁇ l stop solution 95% formamide, 20 mM EDTA xylene cyanol, and bromphenol blue dyes
- an aliquot of the remaining substrate is removed as a zero ribozyme control .
- the samples are separated on either 15% or 20% poly ⁇ acrylamide gels. Each gel is visualized and quantitated with an Ambis beta scanner (Ambis Systems, San Diego, CA) .
- Ambis beta scanner Ambis Systems, San Diego, CA
- kinetic analyses are performed in substrate excess to determine K_. and K cat values.
- the substrates are 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 IX in assay buffer (75 mM Tris- HCl, pH 7.6; 0.1 mM EDTA; 10 mM MgCl 2 ) and contains 58 nanomolar concentration of the long RNA molecules .
- the reaction is started by addition of gel purified ribozymes to 1 ⁇ M concentration. Aliquots are removed at 20, 40, 60, 80 and 100 minutes, then quenched by the addition of 5 ⁇ l stop solution. Cleavage products are separated using denaturing PAGE. The bands are visualized and quantitated with an Ambis beta scanner.
- a simple reaction mechanism for ribozyme-mediated cleavage is : k x k 2
- the rate of the reaction is the rate of disappearance of substrate with time:
- Such ribozymes can be selected and tested in in vivo or ex vivo systems.
- Lipid molecules were dissolved in a volatile organic solvent (CHC1 3 , methanol, diethylether, ethanol, etc.) . The organic solvent was removed by evaporation. The lipid was hydrated into suspension with 0.Ix 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 ⁇ m, 0.2 ⁇ m and 0.1 ⁇ m polycarbonate filters at maximum pressure of 800 psi . The 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.
- CHC1 3 volatile organic solvent
- the suspension was 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 lxPBS and re-extruded through 0.4 ⁇ m, 0.2 ⁇ m, 0.1 ⁇ m polycarbonate filters.
- the liposome entrapped ribozyme was separated from untrapped ribozyme by gel filtration chromatography (SEPHAROSE CL-4B, BIOGEL A5M) .
- the liposome extractions were pooled and sterilized by filtration through a 0.2 ⁇ m filter.
- the free ribozyme was pooled and recovered by ethanol precipitation.
- the liposome concentration was determined by incorporation of a radioactive lipid.
- ribozyme concentration was determined by labeling with 32 P. Rossi et al . , 1992, supra (and references cited therein) , describe other methods suitable for preparation of liposomes .
- a liposome formulation composed of a synthetic lipid derivative disteraoyl-phosphatidyl- ethylamidothioacetyl 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 (calcein was used to follow liposome uptake) . No cytotoxicity was
- liposomes are particularly useful for delivery to CD4 cell populations.
- 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 96 well tissue culture plates. Prior to infection with HIV, cultures are treated for 3 to 24 hours with ribozyme- containing liposomes. Cells are then rinsed with phos ⁇ phate buffered saline (PBS) and virus added at a multi ⁇ 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.
- PBS phos ⁇ phate buffered saline
- 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 incubation at -70°C and 37°C for 30 minutes at each temperature, and viral titers determined by plaque assay using established procedures.
- the accumulation of target mRNA in cells or the cleavage of the RNA by ribozymes or RNaseH can be quantified using an RNase protection assay.
- antisense riboprobes are transcribed from template DNA using T7 RNA polymerase (U.S. Biochemical) in 20 ⁇ l reactions containing IX transcrip ⁇ tion buffer (supplied by the manufacturer) , 0.2 mM ATP, GTP and UTP, 1 U/ ⁇ l pancreatic RNase inhibitor (Boehringer Mannheim Biochemicals) and 200 ⁇ Ci 32 P-labeled CTP (800 Ci/mmol, New England Nuclear) for 1 hour at 37°C.
- Template DNA is digested with 1 U RNase-free DNasel (U.S. Biochemical, Cleveland, OH) at 37°C for 15 minutes and unincorporated nucleotides removed by G-50 SEPHADEX spin chromatography.
- the target RNA can be transcribed in vi tro using a suitable DNA template.
- the transcript is purified by standard methods and digested with ribozyme at 37°C according to methods described later.
- 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 ⁇ l of hybridization buffer (4 M guanidine isothiocyanate, 0.1% sarcosyl, 25 mM sodium citrate, pH 7.5) .
- hybridization buffer 4 M guanidine isothiocyanate, 0.1% sarcosyl, 25 mM sodium citrate, pH 7.5
- Cell lysate 45 ⁇ l or defined amounts of in vi tro transcript (also in hybridization buffer) is then combined with 5 ⁇ l of hybridization buffer containing 5 x 10 5 cpm of each antisense riboprobe in 0.5 ml Eppendorf tubes, over ⁇ laid with 25 ⁇ l mineral oil, and hybridization accomp ⁇ lished by heating overnight at 55°C.
- hybridization reactions are diluted into 0.5 ml RNase solution (20 U/ml RNaseA, 2 U/ml RNaseTl, 10 U/ml RNase-free DNasel in 0.4 M NaCl) , heated for 30 minutes at 37°C, and 10 ⁇ l of 20% SDS and 10 ⁇ l of Proteinase K (10 mg/ml) added, followed by an additional 30 minutes incubation at 37°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°C) in a microcentrifuge.
- Pellets are dissolved in 10 ⁇ l loading buffer (95% formamide, IX TBE, 0.1% bromophenol blue, 0.1% xylene cylanol) , heated to 95°C for five min- utes, cooled on ice, and analyzed on 4% polyacrylamide/7 M urea gels under denaturing conditions.
- 10 ⁇ l loading buffer 95% formamide, IX TBE, 0.1% bromophenol blue, 0.1% xylene cylanol
- 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.
- a reaction mixture contains 1 to 5 pmoles of 5' (kinased) and/or 3' labeled ribozyme, 15 ⁇ g of cytosolic extract and 2.5 mM MgCl 2 in a total volume of 100 ⁇ l. The reaction is incubated at 37°C.
- a 3 '-labeled ribozyme can be formed by incorporation of the 32 P-labeled cordycepin at the 3' OH using poly(A) polymerase.
- the poly(A) polymerase reaction contains 40 mM Tris, pH 8, 10 mM MgCl 2 , 250 mM NaCl, 2.5 mM MnCl 2 ; 3 ⁇ l P 32 cordycepin, 500 Ci/mM; and 6 units poly(A) polymerase in a total volume of 50 ⁇ l . The reaction mixture is incubated for 30 minutes at 37°C.
- Liposomes containing distearoylphosphatidyl ethanol- amidomethyl 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 surface properties of the liposomes. Reaction with N-ethylmaleimide leads to lymphocyte and macrophage uptake. This modification results in liposome uptake by
- CD4 and CD4 + lymphocytes.
- the uptake was assayed in lymphoblasts which had been stimulated for 72 hours with phytohemagglutinin.
- 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.
- 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 HIV-1-homologous substrates.
- the assays are performed in ribozyme excess and approximate K ⁇ /K ⁇ values obtained. Comparison of values obtained with short and long substrates indicates utility in vivo of a ribozyme.
- Ribozymes are 32 P-end- labeled, entrapped in liposomes and delivered to HIV-1 sensitive cells for three hours. The cells are fraction ⁇ ated and ribozyme is purified by phenol/chloroform extraction. Alternatively, cells (lxlO 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, 1 mM EDTA) .
- TSE 10 mM Tris, pH 7.4, 0.25 M Sucrose, 1 mM EDTA
- Nuclei are pelleted at lOOxg 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 7.4, 2.5 mM MgCl 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 ⁇ l 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.
- the cells may be fractioned as above and the purity of each fraction assessed by assaying enzyme activity known to exist in that fraction.
- Ribozyme molecules may be synthesized with 5 phosphorothioate (ps) , or 2'-0-methyl
- 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.
- HeLa (CD4 + ) and Vero cells are used by many ban- gators to screen antiviral chemicals in pre-clinical trials.
- Ribozymes and antisense DNAs depend upon their nuclease stability to maintain therapeutic 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/mg protein) and cation optima (Mg +2 -, Mn +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 levels, and - indicates no nuclease activity as tested by standard methodology) , but the nuclease population present in HeLa cells was dissimilar to that observed in both monocyte 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) .
- HeLa nucleases were maximally active in the presence of added Zn +2 , while added cation did not enhance nuclease activities in monocyte and lymphocyte lysates.
- the rela ⁇ tive nuclease levels in the monocyte and lymphocyte lysates were 2.2 and 4 times higher, respectively, than in activated HeLa lysates.
- ribozymes were tested for their ability to increase nuclease activity while maintaining catalytic activity of the ribozyme.
- Fig. 7 shows the effects of various modifications upon the catalytic activity of a selected ribozyme.
- the modifica ⁇ tions which decreased activity 1315, 1371, and 1285 were dropped from further analyses.
- Chemical modifications to ribozymes resulted in cell-specific patterns of ribozyme stability, and 2'-0-methyl sugar substitution gave the best overall enhancement of ribozyme stability across lysates. Relative resistance of ribozymes to digestion by lymphocyte and monocyte cytoplasmic lysates are shown in Fig. 8.
- the digestion rates of the ribozymes are similar when the ribozymes are labeled at either the 5'- or 3'- ends of the molecule, demonstrating that the degradation is not due to endogenous phosphatase activity in the lysates.
- the 5' exon of tat contains the following potential cleavage sites: 2 GUC sites, 3 GUA sites, 5 AUC sites, 3 UCC sites, and 5 CUC sites. All 18 sites were examined by computer folding and by RNaseH cleavage assay.
- ribozymes are shown in Figs. 3A, B and C, labeled respectively HDH, HEH and HFH.
- the purpose of this example was to evaluate the activity of ribozymes containing substitutions of thiophosphate for phosphate at some backbone positions.
- Ribozymes to the HIV-1 tat gene were synthesized on an ABI synthesizer using standard phosphoramidite chem ⁇ istry, however, at steps where thiophosphate was to be incorporated in the backbone the standard oxidation step
- RNA was gel purified, eluted, kinased and sequenced to ensure that the sequence was correct.
- the end-labeled RNA was also treated with H 2 0 2 which preferentially promotes cleavage at positions containing thiophosphate.
- Ribozyme activity was tested against cleavage of a short (12 nucleotides) end-labeled substrate RNA. Substrate concentration was approximately 1 nM; ribozyme concentration was 5-100 nM; incubation was at 37°C in 75 mM Tris (pH 7.5) , 0.1 mM EDTA, 10 mM MgCl 2 for 2-40 minutes. Cleavage extents were determined by gel electro ⁇ phoresis (PAGE) followed by quantitation on the AMBIS. The ribozymes are shown in Figs. 4A - 4G.
- Ribozymes showed essentially 100% activity: r37 (unmodified) , r37s2 (1 thio on 5' arm, 2 on 3' arm) , r37s4 (3 thio modifications on each arm) , s37A (3 modifications on 3' arm) , s37B (3 modifications alternating on 3' arm) .
- Ribozymes that are fully modified showed some decrease in activity, for example, s37C (almost completely modified on substrate binding arms) showed a 3x reduction in activity relative to unmodified, s37D (thio modifica ⁇ tions in stem I, II and III) showed about a 9x reduction in activity relative to unmodified.
- s37C almost completely modified on substrate binding arms
- s37D thio modifica ⁇ tions in stem I, II and III
- Ribozymes were made on an ABI synthesizer using standard phosphoramidite chemistry with 2'-O-methyl phosphoramidite nucleotides used in place of standard nucleotides. Ribozyme activity was determined in the manner described above with PAGE analysis.
- a ribozyme containing 2'-0- methyl at all positions that base-pair with substrate was synthe- sized.
- the Kcat/Km for the 2' -O-methyl ribozyme was 44 x 10 6 M" 1 min "1 compared to 41 x 10 6 m "1 min "1 for the unmodified, and 32 x 10 6 for a thiophosphate modified ribozyme.
- the 2' -O-methyl ribozyme retains 100% activity.
- the LTR is among the most conserved regions within the HIV-1 genome. Also, the LTR region is present in all the transcripts generated during HIV-1 life cycle. So, ribozymes that cleave LTR targets will potentially block
- HH hammerhead
- Ribozymes with 7/7 binding arms were synthesized using RNA phosphoramadite chemistry. Ribozymes were deprotected and purified as described above.
- Target RNA used in this study was 613 nt long and contained cleavage sites for all the 10 HH ribozymes targeted against LTR.
- a template containing T7 RNA polymerase promoter upstream of LTR target sequence was PCR amplified from an HIV-1 pro-DNA clone. Other such clones can be readily constructed.
- Target RNA was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [ ⁇ .- 32 P] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-1, following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription.
- RNA was precipitated with isopropanol and the pellet was washed two times with 70% ethanol to get rid of salt and nucleotides used in the transcription reaction. RNA is resuspended in ⁇ DEPC-treated water and stored at 4°C.
- Ribozyme Cleavage Reactions Reactions were carried out under ribozyme excess (k cat /Kj conditions (Herschlag and Cech (1990) Biochemistry 29, 10159-10171) . Briefly, 1,000 nM ribozyme and 10 nM internally labeled target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM MgCl 2 . The RNAs were renatured by cooling to 37°C for 10-20 min. Cleavage reaction was initiated by mixing the ribozyme and target RNA at 37°C. Aliquots of 5 ⁇ l were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer and freezing on dry ice. The samples were resolved on sequencing gel.
- HH ribozymes As shown in Fig. 10, of the ten ribozymes that were tested individually, only four HH ribozymes (568, 581, 631, 696) cleaved the LTR RNA. Other target sites appear to be inaccessible to ribozyme binding and cleavage. Site 568 is also referred to as site 115 and has been targeted for hammerhead ribozyme cleavage by Drouplic et al . , 66 J ⁇ . Virol . 1432-1441, 1992 and Heidenreich and Eckstein, 267 J. Biol. Chem. 1904-1909, 1992.
- the 568 HH ribozyme could be stabilized by chemical modification of the sugar moeity of various bases (similar to the ones listed above) .
- the 568 HH ribozyme was extensively modified with one of the following compounds: 2'flouro, 2'amino at the U7 position, araflouro at U4 position, 2'amino at U4 and U7 positions, c-allyl at U4 position. None of the above modifications had any deleterious effect on the ribozyme activity.
- TAT region for HH Ribozyme Sites A region of the TAT mRNA is present in the majority of the transcripts generated during HIV-1 life cycle. So, ribozymes that cleave TAT targets will potentially block HIV replication.
- RNA synthesis and ribozyme cleavage reactions were carried out as described above.
- Target RNA used in this study was 422 nt long.
- Selected ribozymes can be administered prophylactic- ally, or to HIV-1 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 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 unmodified 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 ribozyme uptake, regardless of the delivery vehicle or strategy. Such assays will also determine the intracellular local ⁇ ization 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 include: a. encapsulation in liposomes, b. transduction by retroviral vectors, 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 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.
- the ribozyme may be modified essentially at random, in ways which reduce 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 function ⁇ ally more complex than small drug molecules.
- the struc- tural 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.
- 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.
- 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.
- 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.
- an anti ⁇ body 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 intemalization of the liposomes by the infected cells. Triggering the endocytosis is useful for viral inhibition.
- liposome delivered synthetic 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 smaller ribozymes (less than 142 nucleotides in length) exhibit different intracellular localizations.
- 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.
- Nonoparticles and hydrogels may be potential delivery vehicles for a ribozyme.
- These carriers have been devel ⁇ oped 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.
- permeability enhancers such as Azone or oleic acid
- the liposomes can either represent a slow release presen ⁇ tation vehicle in which the modified ribozyme and perme ⁇ ability enhancer transfer from the liposome into the infected cell, or the liposome phospholipids can partici ⁇ pate directly with the modified ribozyme and permeability enhancer in facilitating cellular delivery.
- 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, intraperi- toneal, 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. This method is particularly useful for treating AIDS using anti-HIV ribozymes of this invention.
- a liposome formulation which can deliver oligonucleotides to lymphocytes and macrophages.
- This oligonucleotide delivery system inhibits HIV proliferation in infected primary immune cells.
- Whole blood studies show that the formulation is taken up by 90% of the lymphocytes after 8 hours at 37°C.
- Preliminary biodistribution and pharmaco- kinetic studies yielded 70% of the injected dose/gm of tissue in the spleen after one hour following intravenous administration.
- This formulation offers an excellent delivery vehicle for anti-AIDS ribozymes for two reasons.
- T-helper lymphocytes and macrophages are the primary cells infected by the virus
- a subcutaneous administration delivers the ribozymes to the resident HIV-infected lymphocytes and macrophages in the lymph node.
- the liposomes then exit the lymphatic system, enter the circulation, and accumulate in the spleen, where the ribozyme is delivered to the resident lymphocytes and macrophages.
- Intraperitoneal administration also leads to entry into the circulation, with once again, the molecular weight or size of the ribozyme-delivery vehicle complex 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 remaining dose circulates in the blood stream for up to 24 hours.
- the chosen method of delivery should result in cytoplasmic accumulation in the afflicted cells 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.
- 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.
- chemically modified ribozymes e.g., with modifica ⁇ tion of the phosphate backbone, or capping of the 5' and 3' ends of the ribozyme with nucleotide analogues 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.
Abstract
Description
Claims
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JP7506461A JPH09501318A (en) | 1993-08-06 | 1994-08-04 | Methods and reagents for inhibiting human immunodeficiency virus replication |
PCT/US1994/008613 WO1995004818A1 (en) | 1993-08-06 | 1994-08-04 | Method and reagent for inhibiting human immunodeficiency virus replication |
EP94922766A EP0716693A1 (en) | 1993-08-06 | 1994-08-04 | Method and reagent for inhibiting human immunodeficiency virus replication |
AU73755/94A AU7375594A (en) | 1993-08-06 | 1994-08-04 | Method and reagent for inhibiting human immunodeficiency virus replication |
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US08/103,423 | 1994-07-07 | ||
PCT/US1994/008613 WO1995004818A1 (en) | 1993-08-06 | 1994-08-04 | Method and reagent for inhibiting human immunodeficiency virus replication |
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WO1997007667A1 (en) * | 1995-08-25 | 1997-03-06 | The Regents Of The University Of California | Ribozymes targetting hiv and siv |
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