US 5,817,796 AGrant
C-Myb Ribozymes Having 2'-5'-Linked Adenylate Residues
Issue Date:1998-10-06
•3 Claims
•24 Drawing Sheets
Abstract
Enzymatic nucleic acid molecules which cleave c-myb RNA or other RNAs associated with restenosis or cancer.
Metadata
Inventors
- Dan T. Stinchcomb
- Kenneth Draper
- James McSwiggen
- Thale Jarvis
Application Information
Application Number:US 4356284
Filing Date:1995-05-05
Priority Date:1992-08-26
Art Unit:189
Classifications
IPC:
C07H 2104C12Q 168
Field of Search:
43553651491.31;91.1;6;172.1;172.323.1;23.2;24.5;25.244
Patent Drawings (24 sheets)
Description
BACKGROUND OF THE INVENTION
The present invention concerns therapeutic compositions and methods for the treatment of restenosis and cancer.
The following is a brief description of the physiology, cellular pathology and treatment of restenosis. The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.
Coronary angioplasty is one of the major surgical treatments for heart disease. Its use has been accelerating rapidly; over 450,000 procedures are performed in the U.S. annually. The short term success rate of angioplasty is 80 to 90%. However, in spite of a number of technical improvements in the procedure, post-operative occlusions of the arteries, or restenosis, still occur. Thirty-five to forty-five percent of patients who have undergone a single vessel angioplasty develop clinically significant restenosis within 6 months of the procedure. The rate of restenosis is even higher (50 to 60%) in patients who have undergone multivessel angioplasty (Califf, R. M., et al., 1990, in Textbook of Interventional Cardiology., E. J. Topol, ed., W. B. Saunders, Philadelphia, pp 363-394.).
Histopathological studies have shown that restenosis after angioplasty is characterized by migration of medial smooth muscle cells to the intima and a striking hyper-proliferative response of these neointimal cells (Garratt, K. N., et al., 1991, J. Am. Coll. Cardio., 17, 442-428; Austin, G. E., et al., 1985, J. Am. Coll. Cardiol., 6, 369-375). Smooth muscle cell proliferation could be an overly robust response to injury. Alternatively, the intimal smooth muscle cells within atherosclerotic lesions are already in an activated or "synthetic" state (Sjolund, M., et al., 1988, J. Cell. Biol., 106, 403-413 and thus may be poised to proliferate. One recent study demonstrated a positive correlation between the presence of activated smooth muscle cells in coronary lesions and the extent of subsequent luminal narrowing after atherectomy (Simons, M., et al., 1993, New Engl. J. Med., 328, 608-613). In any case, slowing smooth muscle cell proliferation after angioplasty could prevent intimal thickening and restenosis.
The presently preferred therapeutic treatment for restenosis is the use of streptokinase, urokinase or other thrombolytic compounds, such as fish oil, anticoagulants, ACE (angiotensin converting enzyme) inhibitors, aspirin and cholesterol lowering compounds. Alternative treatment includes the surgical incorporation of endoluminal stents. The occurrence of pharmacologic side-effects (particularly bleeding disorders associated with anti-coagulants and platelet inhibitors) is an issue with current therapies. Popoma, J. J., et al., report that the current therapies have not significantly impacted the rates of restenosis occurrence.(Circulation, 84, 1426-1436, 1991).
Recently, the results of a clinical trial of the efficacy of an anti-platelet therapy have been reported. Patients undergoing coronary angioplasty were given a single bolus injection followed by a 12 hour infusion of an antibody directed against the platelet adhesion molecule, gpIIb/gpIIIa. After six months, patients with the treatment showed a 23% reduction in the occurrence of restenosis than patients receiving placebo (27 vs. 35%; p=0.001).
A number of growth factors have been shown to induce smooth muscle cell proliferation. In vitro, platelet-derived growth factor (PDGF) is a potent smooth muscle cell mitogen (Ross, R., et al., 1974, Proc. Natl. Acad. Sci. USA, 71, 1207-1210) and a smooth muscle cell chemoattractant (Grotendorst, G., et al., 1982, Proc. Natl. Acad. Sci. USA, 71, 3669-3672.). In vivo, when PDGF is expressed ectopically in porcine arteries, it induces intimal hyperplasia (Nabel, E. B., et al., 1993, J. Clin. Invest., 91, 1822-1829). Furthermore, antibodies to PDGF have been shown to reduce intimal thickening after arterial injury (Ferns, G. A. A., et al., 1991, Science, 253, 1129-1132). Analysis of .sup.3 H-thymidine incorporation in the lesions indicates that the anti-PDGF antibodies primarily inhibit smooth muscle cell migration.
Basic fibroblast growth factor (bFGF) is another smooth muscle cell mitogen in vitro (Klagsbrun, M. and Edelman, E. R., 1989, Arteriosclerosis, 9, 269-278). In a rat model, anti-bFGF antibodies inhibit the proliferation of medial smooth muscle cells 24 to 48 hours after balloon catheter injury (Lidner, V. and Reidy, M. A., 1991, Proc. Natl, Acad. Sci. USA, 88, 3739-3743). In addition to bFGF, heparin binding epidermal growth factor (HB-EGF) (Higashiyama, S., et al., 1991, Science, 251, 936-939.), insulin-like growth factor I (IGF-I) (Banskota, N. K., et al., 1989, Molec. Endocrinol., 3, 1183-1190) and endothelin (Komuro, I., et al., 1988, FEBS Letters, 238, 249-252) have been shown to induce smooth muscle cell proliferation. A number of other factors (such as interleukin-1 and tumor necrosis factor-.alpha.) may indirectly affect smooth muscle cell proliferation by inducing the expression of PDGF (Hajjar, K. A., et al., 1987, J. Exp. Med., 166, 235-245; Raines, E. W., et al., 1989, Science, 243, 393-396).
When whole serum is added to serum-starved smooth muscle cells in vitro, the oncogenes, c-myc, c-fos, and c-myb, are induced (Kindy, M. S. and Sonenshein, G. E., 1986, J. Bio. Chem., 261, 12865-12868; Brown, K. E., et al., 1992, J. Bio. Chem., 267, 4625-4630) and cell proliferation ensues. Blocking c-myb with an antisense oligonucleotide prevents cells from entering S phase (Brown, K. E., et al., 1992, J. Biol. Chem., 267, 4625-4630.). Thus, c-myb is required for the G.sub.1 to S transition after stimulation by the multitude of growth factors present in serum. In vivo, a c-myb antisense oligonucleotide inhibits restenosis when applied to rat arteries after balloon angioplasty (Simons, M., et al., 1992, Nature, 359, 67-70). Similarly, an antisense oligonucleotide directed against mRNA of the oncogene c-myc was shown to inhibit human smooth muscle cell proliferation (Shi, Y., et al., 1993, Circulation, 88, 1190-5) and migration (Biro, S., et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 654-8).
Ohno et al., 1994 Science 265, 781, have shown that a combination of viral thymidine kinase enzyme expression (gene therapy) and treatment with anti-viral drug ganciclovir inhibits smooth muscle cell proliferation in pigs, following baloon angioplasty.
Epstein et al., "Inhibition of non-transformed cell proliferation using antisense oligonucleotides," NTIS publication 1992 discusses use of antisense oligonucleotides to c-myc, PCNA or cyclin B. Fung et al., PCT WO91/15580, describes gene therapy for cell proliferative disease and mentions administration of a ribozyme construct against a PGR element. Mention is made of inactivation of c-myb. Rosenberg et al., WO93/08845, Calabretta et al., WO92/20348 and Gewirtz WO93/09789 concern c-myb antisense oligonucleotides for treatment of melanoma or colorectal cancer, and administration locally. Sytkowski, PCT WO 93/02654, describe the uses of antisense oligonucleotides to inhibit c-myb gene expression in red blood cells to stimulate hemoglobin synthesis.
Nabel and Nabel, U.S. Pat. No. 5,328,470, describe a method for the treatment of diseases by delivering therapeutic reagents directly to the sites of disease. They state that
" . . . Method is based on the delivery of proteins by catheterization to discrete blood vessel segments using genetically modified or normal cells or other vector systems . . . In addition, catalytic RNAs, called ribozymes, can specifically degrade RNA sequences . . . The requirements for a successful RNA cleavage include a hammerhead structure with conserved RNA sequence at the region flanking this structure . . . any GUG sequence within the RNA transcript can serve as a target for degradation by the ribozyme . . . gene transfer using vectors expressing such proteins as tPA for the treatment of thrombosis and restenosis, angiogenesis or growth factors for the purpose of revascularization . . . "
SUMMARY OF THE INVENTION
This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species that are required for cellular growth responses. In particular, applicant describes the selection and function of ribozymes capable of cleaving RNA encoded by the oncogene, c-myb. Such ribozymes may be used to inhibit the hyper-proliferation of smooth muscle cells in restenosis and of tumor cells in numerous cancers. To block restenosis, a target molecule required for the induction of smooth muscle cell proliferation by a number of different growth factors is preferred. To this end c-myc, c-fos, and c-myb are useful targets in this invention.
Other transcription factors involved in the response to growth and proliferation signals include NF-.kappa.B, oct-1 and SRF. NF-.kappa.B protein activates cellular transcription and induces increases in cellular synthetic pathways. In a resting cell, this protein is found in the cytoplasm, complexed with its inhibitor, I-.kappa.B. Upon phosphorylation of the I-.kappa.B molecule, the complex dissociates and NF-.kappa.B is released for transport to the nucleus, where it binds DNA and induces transcriptional activity in (NF-.kappa.B)-responsive genes. One of the (NF-.kappa.B)-responsive genes is the NF-.kappa.B gene itself. Thus, release of the NF-.kappa.B protein from the inhibitory complex results in a cascade of gene expression which is auto-induced. Early inhibition of NF-.kappa.B can reduce expression of a number of genes required for growth and proliferation, such as c-myb.
Two other transcription factors, oct-1 and serum response factor (SRF) have been shown to be expressed selectively in dividing cells. Both oct-1 and SRF are expressed ubiquitously in cultured cells, including smooth muscle cells. However, R. Majack and his colleagues have recently shown that these transcription factors are not expressed by the smooth muscle cells in intact vessels. Both oct-1 and SRF are rapidly expressed upon dispersal of tissue into single cell suspensions. Thus, these transcription factors are thought to be regulated by their interactions with the extracellular matrix (Weiser, M. C. M., et al., 1994, J. Cell. Biochem., S18A, 282; Belknap, J. K., et al., 1994, J. Cell. Biochem., S18A, 277). Upon injury during angioplasty, the expression of oct-1 and SRF may be enhanced, leading to increased smooth muscle cell proliferation. Treatment with ribozymes that block the expression of these transcription factors can alleviate the smooth muscle cell proliferation associated with restenosis.
While some of the above mentioned studies demonstrated that antisense oligonucleotides can efficiently reduce the expression of factors required for smooth muscle cell proliferation, enzymatic RNAs, or ribozymes have yet to be demonstrated to inhibit smooth muscle cell proliferation. Such ribozymes, with their catalytic activity and increased site specificity (as described below), represent more potent and safe therapeutic molecules than antisense oligonucleotides. In the present invention, ribozymes that cleave c-myb mRNA are described. Moreover, applicant shows that these ribozymes are able to inhibit smooth muscle cell proliferation and that the catalytic activity of the ribozymes is required for their inhibitory effect. From those of ordinary skill in the art, it is clear from the examples described, that other ribozymes that cleave target mRNAs required for smooth muscle cell proliferation may be readily designed and are within the invention.
By "inhibit" is meant that the activity of c-myb or level of mRNAs encoded by c-myb is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
By "enzymatic nucleic acid molecule" it is meant a nucleic acid 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 nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic nucleic acid 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. By "equivalent" RNA to c-myb is meant to include those naturally occurring RNA molecules associated with restenosis and cancer in various animals, including human, rat and pig. Such a molecule will generally contain some ribonucleotides, but the other nucleotides may be substituted at the 2'-hydroxyl position and in other locations with other moeities as discussed below.
By "complementarity" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid 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 an enzymatic nucleic acid 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 concentration of ribozyme necessary to affect 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 to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf, T. M., et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al, 1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799) 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 nucleic acid 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 a preferred embodiment the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNAs encoding c-myb proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small enzymatic nucleic acid motifs (e.g., of the hammerhead or the hairpin structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters (e.g., Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992 J. Virol, 66, 1432-41; Weerasinghe et al., 1991 J. Virol, 65, 5531-4; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science 247, 1222-1225). Those skilled in the art realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et al., PCT WO93/23569, and Sullivan et al., PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al., 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994 J. Biol. Chem. 269, 25856)
Thus, in a first aspect, the invention features ribozymes that inhibit cell proliferation. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation is inhibited.
In a preferred embodiment, the enzymatic RNA molecules cleave c-myb mRNA and inhibit smooth muscle cell proliferation. Such ribozymes are useful for the prevention of restenosis after coronary angioplasty. Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. The ribozymes, similarly delivered, also are useful for inhibiting proliferation of certain cancers associated with elevated levels of the c-myb oncogene, particularly leukemias, neuroblastomas, and lung, colon, and breast carcinomas. Using the methods described herein, other enzymatic RNA molecules that cleave c-myb, c-myc, oct-1, SRF, NF-.kappa.B, PDGF receptor, bFGF receptor, angiotensin II, and endothelium-derived relaxing factor and thereby inhibit smooth muscle cell proliferation and/or tumor cell proliferation may be derived and used as described above. Specific examples are provided below in the Tables.
Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the level of c-myb activity in a cell or tissue. By "related" is meant that the inhibition of c-myb mRNAs and thus reduction in the level of protein activity will relieve to some extent the symptoms of the disease or condition.
Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleic acid or nucleic acid, complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers.
In another aspect of the invention, ribozymes that cleave target molecules and inhibit c-myb activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the tables, shown as Seq. I.D. Nos. 1-100. Examples of such ribozymes are shown as Seq. I.D. Nos. 101-129. Those in the art will recognize that while such examples are designed to mouse RNA, similar ribozymes can be made complementary to human RNA. By complementary is thus meant that the binding arms are able to cause cleavage of a human or mouse mRNA target. Examples of such ribozymes consist essentially of sequences defined as Seq. I.D. Nos. 101-129 below. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind human mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
In another aspect of the invention, ribozymes that cleave target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or viral vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells. Once expressed, the ribozymes cleave their target mRNAs and prevent proliferation of their host cells. The recombinant vectors are preferably DNA plasmids or adenovirus vectors. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The drawings will first briefly be described.
Drawings
FIG. 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be .gtoreq.2 base-pair long.
FIG. 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; FIG. 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; FIG. 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and FIG. 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.
FIG. 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is .gtoreq.1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is .gtoreq.2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. ".sub.------ " refers to a covalent bond.
FIG. 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art.
FIG. 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.
FIG. 6 is a schematic representation of an RNAseH accessibility assay. Specifically, the left side of FIG. 6 is a diagram of complementary DNA oligonucleotides bound to accessible sites on the target RNA. Complementary DNA oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is represented by the thin, twisted line. The right side of FIG. 6 is a schematic of a gel separation of uncut target RNA from a cleaved target RNA. Detection of target RNA is by autoradiography of body-labeled, T7 transcript. The bands common to each lane represent uncleaved target RNA; the bands unique to each lane represent the cleaved products.
FIG. 7 is a graph of the results of an RNAseH accessibility assay of murine c-myb RNA. On the abscissa is the sequence number of the DNA oligonucleotide that is homologous to the ribozyme target site. The ordinate represents the percentage of the intact transcript that was cleaved by RNAse H.
FIG. 8 is a graph of the outcome of an RNAseH accessibility assay of human c-myb mRNA. The graphs are labeled as in FIG. 7.
FIG. 9 shows the effect of chemical modifications on the catalytic activity of hammerhead ribozyme targeted to c-myb site 575. A) diagrammatic representation of 575 hammerhead ribozyme.cndot.substrate complex. 2'-O-methyl ribozyme represents a hammerhead (HH) ribozyme containing 2'-O-methyl substitutions at five nucleotides in the 5' and 3' termini. 2'-O-methyl P=S ribozyme represents a hammerhead (HH) ribozyme containing 2'-O-methyl and phosphorothioate substitutions at five nucleotides in the 5' and 3' termini. 2'-C-allyl iT ribozyme represents a hammerhead containing ribose residues at five positions. The remaining 31 nucleotide positions contain 2'-hydroxyl group substitutions, wherein 30 nucleotides contain 2'-O-methyl substitutions and one nucleotide (U.sub.4) contains 2'-C-allyl substitution. Additionally, 3' end of this ribozyme contains a 3'--3' linked inverted T. 2'-C-allyl P=S ribozyme is similar to 2'-C-allyl iT ribozyme with the following changes: five nucleotides at the 5' and 3' termini contain phosphorothioate substitutions and the ribozyme lacks the 3'-end inverted T modification. B) shows the ability of ribozymes described in FIG. 9A to inhibit smooth muscle cell proliferation.
FIG. 10 shows the effect of 2'-C-allyl P=S 575 HH ribozyme concentration on smooth muscle cell proliferation. A plot of percent inhibition of smooth muscle cell proliferation (normalized to the effect of a catalytically inactive ribozyme) as a function of ribozyme concentration is shown.
FIG. 11 shows a comparison of the effects of 2'-C-allyl P=S 575 HH ribozyme and phosphorothioate antisense DNA on the proliferation of smooth muscle cells.
FIG. 12 shows the inhibition of smooth muscle cell proliferation catalyzed by 2'-C-allyl P=S HH ribozymes targeted to sites 549, 575, and 1533 within c-myb mRNA.
FIG. 13 shows the effect of phosphorthioate substitutions on the catalytic activity of 2'-C-allyl 575 HH ribozyme. A) diagrammatic representation of 575 hammerhead ribozyme.cndot.substrate complex. 10 P=S 5' and 3' ribozyme is identical to the 2'-C-allyl P=S ribozyme described in
FIG. 9. 5 P=S 3' ribozyme is same as 10 P=S 5' and 3' ribozyme, with the exception that only five nucleotides at the 3' termini contain phosphorothioate substitutions. 5 P=S Loop ribozyme is similar to 2'-C-allyl iT described in FIG. 9, with the exception that five nucleotides within loop 11 of this ribozyme contain phosphorothioate substitutions. 5 P=S 5' ribozyme is same as 10 P=S 5' and 3' ribozyme, with the exception that only five nucleotides at the 5' termini contain phosphorothioate substitutions. Additionally, this ribozyme contains a 3'--3' linked inverted T at its 3' end. B) shows the ability of ribozymes described in FIG. 13A to inhibit smooth muscle cell proliferation.
FIG. 14 shows the minimum number of phosphorothioate substitutions required at the 5' termini of 575 HH ribozyme to achieve efficient inhibition of smooth muscle cell proliferation.
FIG. 15 shows the effect of varying the length of substrate binding arm of 575 HH ribozyme on the inhibition of smooth muscle cell proliferation.
FIG. 16 shows the effect of various chemical modifications, at U.sub.4 and/or U.sub.7 positions within 575 HH ribozyme core, on the ability of the ribozyme to inhibit smooth muscle cell proliferation.
FIG. 17 shows the inhibition of pig smooth muscle cell proliferation by active c-myb 575 HH ribozyme.
FIG. 18 shows the inhibition of human smooth muscle cell proliferation by active c-myb 575 HH ribozyme.
FIG. 19 shows inhibition of intimal hyperplasia in injured pig vessels by locally delivered synthetic RNA.
FIG. 20 is digrammatic representation of an optimal c-myb HH ribozyme that can be used to treat diseases like restenosis.
FIG. 21 shows the inhibition of Rat smooth muscle cells by 2-5A containing nucleic acids.
Target sites
Targets for useful ribozymes can be determined as disclosed in Draper et al supra. Sullivan et al., supra, as well as by Draper et al., "Method and reagent for treatment of arthritic conditions U.S. Ser. No. 08/152,487, filed Nov. 12, 1993, and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein. While specific examples to mouse RNA are provided, those in the art will recognize that equivalent human RNA targets can be used as described below. Thus, the same target may be used, but binding arms suitable for targetting human RNA sequences are present in the ribozyme. Such targets may also be selected as described below.
The sequence of human, pig and murine c-myb mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables II and IV (All sequences are 5' to 3' in the tables) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. While murine, pig and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, as discussed in Stinchcomb et al., "Method and Composition for Treatment of Restenosis and Cancer Using Ribozymes," filed May 18, 1994, U.S. Ser. No. 08/245,466, murine and pig targeted ribozymes may be useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.
Hammerhead or hairpin ribozymes were designed that could bind and were individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
The sequences of the ribozymes that are chemically synthesized, useful in this study, are shown in Table III. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Table III (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Table III (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Table III may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.
Optimizing Ribozyme Activity
Ribozyme activity can be optimized as described in this application. These include altering the length of the ribozyme binding arms (stems I and III, see FIG. 2c), or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Eckstein et al, International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Usman, N. et al. U.S. patent application Ser. No. 07/829,729, and Sproat, European Patent Application 92110298.4 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules, modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements. (All these publications are hereby incorporated by reference herein.),
Sullivan, et al., supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al., supra and Draper et al, supra which have been incorporated by reference herein.
Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol, 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U.S.A., 90, 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors).
In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves mRNAs encoded by c-myb is inserted into a plasmid DNA vector or an adenovirus or adeno-associated virus DNA viral vector or a retroviral RNA vector. Viral vectors have been used to transfer genes and lead to either transient or long term gene expression (Zabner et al., 1993 Cell 75, 207; Carter, 1992 Curr. Opi. Biotech. 3, 533).
The adenovirus vector is delivered as recombinant adenoviral particles. The DNA may be delivered alone or complexed with vehicles (as described for RNA above). The recombinant adenovirus or AAV particles are locally administered to the site of treatment, e.g., through incubation or inhalation in vivo or by direct application to cells or tissues ex vivo.
In another preferred embodiment, the ribozyme is administered to the site of c-myb expression (e.g., smooth muscle cells) in an appropriate liposomal vesicle.
EXAMPLES
Ability Of Exogenously-Delivered Ribozymes Directed Against c-myb To Inhibit Vascular Smooth Muscle Cell Proliferation
The following examples demonstrate the selection of ribozymes that cleave c-myb mRNA. The methods described herein represent a scheme by which ribozymes may be derived that cleave other mRNA targets required for cell division. Also provided is a description of how such ribozymes may be delivered to smooth muscle cells. The examples demonstrate that upon delivery, the ribozymes inhibit cell proliferation in culture. Moreover, no inhibition is observed if mutated ribozymes that are catalytically inactive are applied to the cells. Thus, inhibition requires the catalytic activity of the ribozymes. The cell division assay used represents a model system for smooth muscle cell hyperproliferation in restenotic lesions.
Example 1
Identification of Potential Ribozyme Cleavage Sites in Human c-myb mRNA
The sequence of human c-myb mRNA was screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and contained potential hammerhead ribozyme cleavage sites were identified. These sites are shown in Table II and are identical to Table I of Draper, "Method and Reagent for Treatment of a Stenotic Condition", U.S. Ser. No. 07/987,132. (All sequences are 5' to 3' in the tables.) In the original, the sites were identified using nucleotide numbers from (Majello, B., et al., 1986, Proc. Natl. Acad. Sci. USA, 83, 9636-9640) (GenBank Accession No. M15024). Here, we report sites using the sequence numbers from (Westin, E. H., et al., 1990, Oncogene, 5, 1117-1124) (GenBank Accession No. X52125); the latter sequence is derived from a longer c-myb cDNA isolate and thus is more representative of the full-length RNA.
Example 2
Selection of Ribozyme Cleavage Sites in Murine and Human c-myb mRNA
To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in c-myb RNA, 41 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by comparing cDNA sequences of mouse and human c-myb (GenBank Accession No. X02774 and GenBank Accession No. X52125, repsectively) and prioritizing the sites on the basis of overall nucleotide sequence homology. Hammerhead ribozymes were designed that could bind each target (see FIG. 2C) and were individually analyzed by computer folding (Jaeger, J. A., et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706-7710) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
Example 3
Screening Ribozyme Cleavage Sites by RNaseH Protection
Murine and human mRNA was screened for accessible cleavage sites by the method described generally in McSwiggen, U.S. patent application Ser. No. 07/883,849 filed May 1, 1992, entitled "Assay for ribozyme target site," hereby incorporated by reference herein. Briefly, DNA oligonucleotides representing 41 potential hammerhead ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from human or murine c-myb cDNA clones. Labeled RNA transcripts were synthesized in vitro from the two templates. The oligonucleotides and the labeled transcripts were annealed, RNAseH was added and the mixtures were incubated for the designated times at 37.degree. C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a phosphor imaging system. The results are shown in FIGS. 7 and 8. From these data, 20 hammerhead ribozyme sites were chosen as the most accessible (see Table III). Eighteen of the twenty sites chosen overlap sequences shown in Table II; thus, the RNA folding is predictive of accessible regions in the RNA.
Example 4
Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of c-myb RNA
Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990 Nucleic Acids Res., 18, 5433 and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields were >98%. Inactive ribozymes were synthesized by substituting a U for G.sub.5 and a U for A.sub.14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). All ribozymes were modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Usman et al., Synthesis, deprotection, analysis and purification of RNA and ribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736 the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table III.
Example 5
Ribozyme Cleavage of Long Substrate RNA Corresponding to c-myb mRNA Target
Hammerhead-type ribozymes which were targeted to the murine c-myb mRNA were designed and synthesized to test the cleavage activity at the 20 most accessible sites in in vitro transcripts of both mouse and human c-myb RNAs. The target sequences and the nucleotide location within the c-myb mRNA are given in Table IV. All hammerhead ribozymes were synthesized with binding arm (Stems I and II; see FIG. 2C) lengths of seven nucleotides. Two hairpin ribozymes were synthesized to sites 1632 and 2231. The relative abilities of these ribozymes to cleave both murine and human RNAs is summarized in Table IV. Ribozymes (1 .mu.M) were incubated with .sup.32 P-labeled substrate RNA (prepared as described in Example 3, approximately 20 nM) for 60 minutes at 37.degree. C. using buffers described previously. Intact RNA and cleavage products were separated by electrophoresis through polyacrylamide gels. The percentage of cleavage was determined by Phosphor Imager.RTM. quantitation of bands representing the intact substrate and the cleavage products.
Five hammerhead ribozymes (directed against sites 549, 575, 1553, 1597, and 1635) and one hairpin ribozyme (directed against site 1632) were very active; they cleaved >70% of both murine and human c-myb RNA in 60 minutes. Nine of the hammerhead ribozymes (directed against sites 551, 634, 936, 1082, 1597, 1721, 1724, 1895, and 1943) were intermediate in activity, cleaving >50% of both murine and human c-myb RNA in 60 minutes. All of the sites cleaved by these active ribozymes were predicted to be accessible to ribozyme cleavage in Table 2. Six hammerhead ribozymes and one hairpin ribozyme showed low activity on at least one of the substrates. The observed differences in accessibility between the two species of c-myb RNA demonstrate the sensitivity of ribozyme action to RNA structure and suggest that even when homologous target sequences exist, ribozymes may be excluded from cleaving that RNA by structural constraints. This level of specificity minimizes non-specific toxicity of ribozymes within cells.
Example 6
Ability of Hammerhead Ribozymes to Inhibit Smooth Muscle Cell Proliferation.
The ribozymes that cleaved c-myb RNA described above were assayed for their effect on smooth muscle cell proliferation. Rat vascular smooth muscle cells were isolated and cultured as follows. Aortas from adult Sprague-Dawley rats were dissected, connective tissue was removed under a dissecting microscope, and 1 mm.sup.2 pieces of the vessel were placed, intimal side up, in a Petri dish in Modified Eagle's Medium (MEM) with the following additives: 10% FBS, 2% tryptose phosphate broth, 1% penicillin/streptomycin and 2 mM L-Glutamine. The smooth muscle cells were allowed to migrate and grow to confluence over a 3-4 week period. These primary cells were frozen and subsequent passages were grown at 37.degree. C. in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and the following additives: 2 mM L-Glutamine, 1% penicillin/streptomycin, 1 mM sodium pyruvate, non-essential amino acids (0.1 mM of each amino acid), and 20 mM Hepes pH 7.4. Cells passed four to six times were used in proliferation assays. For the cell proliferation assays, 24-well tissue culture plates were prepared by coating the wells with 0.2% gelatin and washing once with phosphate-buffered saline (PBS). RASMC were inoculated at 1.times.10.sup.4 cells per well in 1 ml of DMEM plus 10% FBS and additives and incubated for 24 hours. The cells were subconfluent when plated at this density. The cells were serum-starved by removing the medium, washing once with PBS, and incubating 48-72 hours in DMEM containing 0.5% FBS plus additives.
In several other systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, C. F., et al., 1992, Mol. Pharmacology, 41, 1023-1033). In many of the following experiments, ribozymes were complexed with cationic lipids. The cationic lipid, Lipofectamine (a 3:1 (w/w) formulation of DOSPA (2,3-dioleyloxy-N-2(sperminecarboxamido)ethyl!-N,N-dimethyl-1-propanamini um trifluoroacetate) and dioleoyl phosphatidylethanolamine (DOPE)), was purchased from Life Technologies, Inc. DMRIE (N-1-(2, 3-ditetradecyloxy)propyl!-N,N-dimethyl-N-hydroxyethylammonium bromide) was obtained from VICAL. DMRIE was resuspended in CHCl.sub.3 and mixed at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). The CHCl.sub.3 was evaporated, the lipid was resuspended in water, vortexed for 1 minute and bath sonicated for 5 minutes. Ribozyme and cationic lipid mixtures were prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives was warmed to room temperature (about 20-25.degree. C.), cationic lipid was added to the final desired concentration and the solution was vortexed briefly. RNA oligonucleotides were added to the final desired concentration and the solution was again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex was serially diluted into DMEM following the 10 minute incubation.
Serum-starved smooth muscle cells were washed twice with PBS, and the RNA/lipid complex was added. The plates were incubated for 4 hours at 37.degree. C. The medium was then removed and DMEM containing 10% FBS, additives and 10 .mu.M bromodeoxyuridine (BrdU) was added. In some wells, FBS was omitted to determine the baseline of unstimulated proliferation. The plates were incubated at 37.degree. C. for 20-24 hours, fixed with 0.3% H.sub.2 O.sub.2 in 100% methanol, and stained for BrdU incorporation by standard methods. In this procedure, cells that have proliferated and incorporated BrdU stain brown; non-proliferating cells are counter-stained a light purple. Both BrdU positive and BrdU negative cells were counted under the microscope. 300-600 total cells per well were counted. In the following experiments, the percentage of the total cells that have incorporated BrdU (% cell proliferation) is presented. Errors represent the range of duplicate wells. Percent inhibition then is calculated from the % cell proliferation values as follows: % inhibition=100-100((Ribozyme -0% serum)/(Control-0% serum)).
Six hammerhead ribozymes, including the best five ribozymes from the in vitro RNA cleavage test (directed against sites 549, 575, 1553, 1598, and 1635) and one with intermediate cleavage levels (directed against site 1597) and their catalytically inactive controls were synthesized and purified as described above. The ribozymes were delivered at a concentration of 0.3 .mu.M, complexed with DMRIE/DOPE such that the cationic lipid charges and the anionic RNA charges were at 1:1 molar ratio. The results, shown in Table V, demonstrate a considerable range in the efficacy of ribozymes directed against different sites. Five of the six hammerhead ribozymes (directed against sites 549, 575, 1553, 1597, and 1598) significantly inhibit smooth muscle cell proliferation. The control, inactive ribozymes that cannot cleave c-myb RNA due to alterations in their catalytic core sequence fail to inhibit rat smooth muscle cell proliferation. Thus, inhibition of cell proliferation by these five hammerhead sequences is due to their ability to cleave c-myb RNA, and not because of any antisense activity. The sixth ribozyme (directed against site 1635) fails to function in smooth muscle cells. This ribozyme cleaved c-myb RNA very efficiently in vitro. In this experiment, 10% FBS (no ribozyme added) induced 64.+-.1% proliferation; 0% FBS produced a background of 9.+-.1% proliferation.
Example 7
Ability of exogenously delivered hairpin ribozyme against c-myb to inhibit vascular smooth muscle cell proliferation
In addition to the hammerhead ribozymes tested above, a bipartite hairpin ribozyme (Chowrira, B. M., supra, 1992, Nucleic Acids Res., 20, 2835-2840) was identified that also cleaves c-myb RNA. The effect of this ribozyme on smooth muscle cell proliferation was tested. Ribozymes were delivered at the indicated doses with Lipofectamine at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) induced 87.+-.1%. proliferation; 0% FBS produced 5.+-.1% proliferation. The results of a dose-response experiment are shown in Table VI. In this example, the control was an irrelevant hammerhead ribozyme. The irrelevant ribozyme control contains the same catalytic core sequences, but has binding arms that are directed to a cellular RNA that is not required for smooth muscle cell proliferation. This control failed to significantly inhibit cell proliferation, demonstrating the sequence specificity of these ribozymes. Another control that could be run is an irrelevant catalytically active ribozyme having the same GC content as the test ribozyme.
Example 8
Ribozymes inhibit proliferation of rat smooth muscle cells in a dose-dependent fashion.
If the inhibition of proliferation observed in Example 6 is caused by the ribozymes, the level of inhibition should be proportional to the dose of RNA added. Rat aortic smooth muscle cells were assayed for proliferation in the presence of differing doses of two hammerhead ribozymes. The results shown in Table VII indicate that two hammerhead ribozymes that cleave c-myb RNA at sites 575 and 549 inhibit SMC proliferation in a dose-dependent fashion. Ribozymes were delivered with the cationic lipid, Lipofectamine at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) gave 92 i 1% proliferation; 0% FBS gave 6.+-.1% proliferation. The control is an active ribozyme directed against an irrelevant mRNA target and shows no inhibition over the dose range tested. The control ribozyme contains the same catalytic core sequences as the active ribozymes but differs in its binding arm sequences (stems I and III in FIG. 2c). Thus, ribozyme inhibition of smooth muscle cell proliferation requires sequence-specific binding by the hammerhead arms to c-myb mRNA.
Example 9
Delivery of a c-myb Ribozyme With Different Cationic Lipids
The experiment in Table VIII shows the response of rat smooth muscle cells to a hammerhead ribozyme that cleaves c-myb RNA at site 575 delivered with two different cationic lipids, DMRIE and Lipofectamine.
Similar efficacy is observed with either lipid. 10% FBS (no ribozyme) induced 78.+-.2% proliferation; 0% FBS produced a background of 6.+-.1% proliferation.
Example 10
Effect of varying arm-lengths on ribozyme activity.
The exact configuration of each ribozyme can be optimized by altering the length of the binding arms (stems I and III, see FIG. 2C). The length of the binding arms may have an effect on both the binding and the catalytic cleavage step (Herschlag, D., 1991, Proc. Natl. Acad. Sci. USA, 88, 6921-5). For example, Table IX shows the ability of arm length variants of c-myb hammerhead 575 to inhibit SMC proliferation. Note that the dose used in this experiment (0.1 .mu.M) is 3-fold lower than in previous experiments. At this concentration, the 7/7 arm variant gives relatively little inhibition. In this case, the degree of inhibition increases with concomitant increases in arm length.
The optimum arm length may be site-specific and should be determined empirically for each ribozyme. Towards this end, hammerhead ribozymes target with 7 nucleotide binding arms (7/7) and ribozymes with 12 nucleotide binding arms (12/12) targeted to three different cleavage sites were compared.
Ribozymes were delivered at 0.2 .mu.M with the cationic lipid DMRIE at a 1:1 charge ratio of oligonucleotide to cationic lipid as described in Example 6. The data are shown below in Table X. As can be seen, all three ribozymes demonstrated enhanced inhibition of smooth muscle cell proliferation with twelve nucleotide binding arms. Each ribozyme showed greater inhibition than its catalytically inactive control, again demonstrating that the ribozymes function via their ability to cleave c-myb RNA. In this experiment, 10% stimulation resulted in 54.+-.2% cell proliferation;
unstimulated cells showed 8.+-.0.5% cell proliferation.
Example 11
Effect of chloroquine on ribozyme activity.
A number of substances that effect the trafficking of macromolecules through the endosome have been shown to enhance the efficacy of DNA delivery to cells. These include, but are not limited to, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), chloroquine, monensin, colchicine, and viral particles (Cotten, M. et al., 1990, Proc. Natl. Acad. Sci. USA, 87, 4033-4037; Cotten, M. et al., 1993, J. Virol., 67, 3777-3785; Cotten, M. et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 6094-6098; Cristiano, R. J. et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 2122-6; Curiel, D. T. et al., 1991, Proc. Nat. Acad. Sci. USA, 88, 8850-8854; Ege, T. et al., 1984, Exp. Cell Res., 155, 9-16; Harris, C. E. et al., 1993, Am. J. Respir. Cell Mol. Biol., 9, 441-7; Seth, P. et al., 1994, J. Virol., 68, 933-40; Zenke, M. et al., 1990, Proc. Natl. Acad. Sci. USA, 87, 3655-3659). It is thought that DNA is taken up by cells by endocytosis, resulting in DNA accumulation in endosomes (Akhtar, S. and Juliano, R. L., 1992, Trends Cell Biol., 2, 139-144). Thus, the above agents may enhance DNA expression by promoting DNA release from endosomes. To determine whether such agents may augment the functional delivery of RNA and ribozymes to smooth muscle cells, the effects of chloroquine on ribozyme inhibition of smooth muscle cell proliferation were assessed. A ribozyme with twelve nucleotide binding arms that cleaves c-mby RNA was delivered to rat smooth muscle cells as described in Example 6 (0.2 .mu.M ribozyme complexed with DMRIE/DOPE at a 1:1 charge ratio). In some cases, 10 .mu.M chloroquine was added upon stimulation of the cells. The addition of choloroquine had no effect on untreated cells (stimulation with 10% serum in the presence or absence of chloroquine resulted in 80.5.+-.1.5% and 83.+-.2% cell proliferation, respectively; unstimulated cells with and without chloroquine showed 7.+-.0.5% and 7.+-.1% cell proliferation, respectively). As shown in Table Xl below, addition of chloroquine augments ribozyme inhibition of smooth muscle cell proliferation two- to three-fold.
Example 12
Effect of a hammerhead ribozyme on human smooth muscle cell proliferation.
The hammerhead ribozyme that cleaves human c-myb RNA at site 549 was tested for its ability to inhibit human aortic smooth muscle cell proliferation. The binding site for this ribozyme is completely conserved between the mouse and human cDNA sequences. Human aortic smooth muscle cells (AOSMC) were obtained from Clonetics and were grown in SmGM (Clonetics.RTM.). Cells from passage five or six were used for assays. Conditions for the proliferation assay were the same as for the rat cells (see Example 6), except that the cells were plated in SmGM and starved in SmBM plus 0.5% FBS. The ribozyme that cleaves site 549 was delivered at varying doses complexed with the cationic lipid DMRIE at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) induced 57.+-.7% proliferation; the uninduced background was 6.+-.1% proliferation. The results in Table XII show that inhibition is observed over a similar concentration range as was seen with rat smooth muscle cells.
Example 13
Inhibition by direct addition of a modified, stabilized ribozyme.
A hammerhead ribozyme that cleaves site 575 was chemically synthesized with 12 nucleotide binding arms (sequence ID NO. 127, in Table III). Chemically modified nucleotides were incorporated into this ribozyme that have been shown to enhance ribozyme stability in serum without greatly impacting catalytic activity. (See Eckstein et al., International Publication No. WO 92/07065, Perrault et al., 1990, Nature, 344, 565-568, Pieken, W. et al. 1991, Science, 253, 314-317, Usman, N.; Cedergren, R. J., 1992, Trends in Biochem. Sci., 17, 334-339, Usman, N. et al. U.S. patent application Ser. No. 07/829,729 and Sproat, B. European Patent Application 92110298.4 describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.) The modifications used were as follows. All the nucleotides of the ribozyme contained 2'-O-methyl groups with the following exceptions: U.sub.4 and U.sub.7 contained 2'-amino substitutions; G.sub.5, A.sub.6, G.sub.8, G.sub.12, and A.sub.15.1 were 2'-OH ribonucleotides (numbering as in FIG. 1). An inactive ribozyme was chemically synthesized in which G.sub.5 and A.sub.14 were substituted with 2'-O-methyl U. Ribozymes were added to rat smooth muscle cells at the indicated concentrations as per Example 6 except that cationic lipids were omitted. Proliferation was assessed by BrdU incorporation and staining. Table XII shows that the modified ribozyme is capable of inhibiting rat smooth muscle cell proliferation without addition of cationic lipids. In this experiment, 10% serum induced 45.+-.2% proliferation while uninduced cells showed a background of 2.3.+-.0.1% proliferation.
Optimizing Ribozyme Activity
As demonstrated in the above examples, ribozymes that cleave c-myb RNA are capable of inhibiting 50% of the smooth muscle cells from proliferating in response to serum. This level of inhibition does not represent the maximal effect obtainable with the ribozymes; in each dose response experiment, the highest dose produced the greatest extent of inhibition. Thus, optimizing activity of the ribozyme within the cells and/or optimizing the delivery of the ribozyme to the cells is expected to increase the extent of inhibition.
Tables IX and X demonstrate one means of optimizing ribozyme activity. By altering the length of the ribozyme binding arms (stems I and III, see FIG. 2c), the ability of the ribozyme to inhibit smooth muscle cell proliferation is greatly enhanced. Ribozymes with increasing arm lengths will be synthesized either chemically in one or two parts (see above and see Mamone, U.S. Ser. No. 07/882,689, filed May 11, 1992, hereby incorporated by reference herein) or by in vitro transcription (see Cech et al., U.S. Pat. No. 4,987,071). Ribozymes are chemically synthesized with modifications that prevent their degradation by serum ribonucleases (as described in Example 13, above). When synthesized in two parts, the fragments are ligated or otherwise juxtaposed as described (see original application and Mamone, supra). The effects of the ribozymes on smooth muscle cell proliferation are assessed as in Examples 6 and 12, above. As the length of stems I and III can affect both hybridization to the target and the catalytic rate, the arm length of each ribozyme will be optimized for maximal inhibitory effect in cells. Similarly, the precise sequence of modified nucleotides in the stabilized ribozyme will affect the activity in cells. The nature of the stabilizing modifications will be optimized for maximal inhibitory effect in cells. In each case, activity of the ribozyme that cleaves c-myb RNA will be compared to the activity of its catalytically inactive control (substitution of 2'-O-methyl U for G.sub.5 and a 2'-O-methyl U for A.sub.14) and to a ribozyme targeted to an irrelevant RNA (same catalytic core, with appropriate modifications, but different binding arm sequences).
Sullivan, et al., supra, describes the general methods for delivery of enzymatic RNA molecules. The data presented in Example 9 indicate that different cationic lipids can deliver active ribozymes to rat smooth muscle cells. In this example, 0.6 .mu.M ribozyme delivered with Lipofectamine produced the same inhibitory effect as 0.3 .mu.M ribozyme delivered with DMRIE. Thus, DMRIE is twice as efficacious as Lipofectamine at delivering active ribozymes to smooth muscle cells. There are a number of other cationic lipids known to those skilled in the art that can be used to deliver nucleic acid to cells, including but not limited to dioctadecylamidoglycylspermine (DOGS), dioleoxltrimetylammonium propane (DOTAP), N-1-(2,3-dioleoyloxy)-propyl!-n,n,n-trimethylammoniumchloride (DOTMA), N-1-(2,3-dioleoyloxy)-propyl!-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), and N-1-(2,3-dioleoyloxy)propyl!-N,N-dimethyl-N-hydroxypropylammonium bromide (DORIE-HP). Experiments similar to those performed in Example 9 are used to determine which lipids give optimal delivery of ribozymes to smooth muscle cells. Other such delivery methods are known in the art and can be utilized in this invention.
The data described in Example 11 show that ribozyme delivery and efficacy may be augmented by agents that disrupt or alter cellular endosome metabolism. Chloroquine was shown to increase the ability of a ribozyme to inhibit smooth muscle cell proliferation by 2- to 3-fold. Experiments similar to those described in Example 11 can be performed to determine the optimal concentration of chloroquine to be used to augment delivery of ribozymes alone (as in Example 13), or delivery in the presence different cationic lipids (as in Example 9 and described above) or with other delivery agents (as described below). Other agents that disrupt or alter endosomes known to those familiar with the art can be used to similarly augment ribozyme effects. These agents may include, but are not limited to, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), chloroquine, monensin, colchicine, amphipathic peptides, viral proteins, and viral particles. Such compounds may be used in conjunction with ribozymes as described above, may be chemically conjugated directly to ribozymes may be chemically conjugated to liposomes, or may be incorporated with ribozymes in liposome particles (see Sullivan, et al, supra, incorporated by reference herein).
The data presented in Example 13 indicate that the proliferation of smooth muscle cells can be inhibited by the direct addition of chemically stabilized ribozymes. Presumably, uptake is mediated by passive diffusion of the anionic nucleic acid across the cell membrane. In this case, efficacy could be greatly enhanced by directly coupling a ligand to the ribozyme. The ribozymes are then delivered to the cells by receptor-mediated uptake. Using such conjugated adducts, cellular uptake can be increased by several orders of magnitude without having to alter the phosphodiester linkages necessary for ribozyme cleavage activity.
Alternatively, ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Alternative routes of delivery include, but are not limited to, intramuscular injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan, et al., supra and Draper, et al., supra which have been incorporated by reference herein.
Example 14
Phosphorothioate linkages enhance the ability of ribozymes to inhibit smooth muscle cell proliferation.
As the applicant had shown in Example 13, the hammerhead (HH) ribozyme that cleaves c-myb RNA at site 575 can be modified to confer resistance to nucleases while maintaining catalytic activity (see also Usman et al., supra). To identify ribozymes with optimal activity in cells, several different chemically-modified ribozymes were directly compared for inhibition of rat smooth muscle cell proliferation. Chemically-modified ribozymes used are diagrammed in FIG. 9A. One ribozyme (designated "2'-O-methyl") contains ribonucleotide residues at all positions except the 5 terminal nucleotides of each target binding arm (Stems I and III). The ribozyme designated "2'-O-methyl P=S" in addition contains five phosphorothioate linkages between the terminal nucleotides in each target binding arm. The ribozyme termed "2'-C-allyl iT" contains thirty 2'-O-methyl nucleotides as specified in Example 13. The ribozyme also contains 2'-C-allyl U (Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163) at the U4 position and 2'-O-methyl U at the U7 position and a 3'--3'-linked inverted thymidine (Ortigao et al., 1992 Antisense Res. & Development 2, 129; Seliger et al., Canadian Patent Application No. 2,106,819) at the 3' end of the molecule (referred to as 2'-C-allyl iT). The fourth ribozyme contains the same 2'-O-methyl and 2'-C-allyl residues described above with the addition of 5 phosphorothioate linkages between the terminal nucleotides in each target binding arm (referred to as "2'-C-allyl P=S").
Ribozymes were delivered to smooth muscle cells as cationic lipid complexes (Sullivan et al., supra). In this example, the cationic lipid, Lipofectamine (GIBCO-BRL), was used at a charged lipid concentration of 3.6 .mu.M (see Examples 6 and 9). Active versus inactive forms of each ribozyme were compared to determined whether inhibition is mediated specifically by ribozyme cleavage. As shown in FIG. 9B, the ribozyme synthesized with the 2'-C-allyl modification and the phosphorothioate linkages demonstrated enhanced inhibition of smooth muscle cell proliferation. The catalytically inactive form of the ribozyme had little effect on cell proliferation; thus, the inhibition observed requires the catalytic activity of the ribozyme. In contrast, ribozymes without the stable 2'-O-methyl- and 2'-C-allyl-modified catalytic core (2'-O-methyl and 2'-O-methyl P=S) at best showed only modest inhibition of smooth muscle cell proliferation. The stable core chemistry alone was not sufficient to greatly enhance ribozyme-mediated inhibition; without terminal P=S linkages, the 2'-C-allyl-modified ribozyme showed very little specific inhibition when compared to its inactive ribozyme control. These results demonstrate that certain chemical modifications greatly enhance the ability of exogenously-delivered ribozymes to cleave c-myb RNA and impact cell proliferation.
Example 15
Dose response of the chemically modified ribozyme.
Varying doses of the 2'-C-allyl P=S-modified ribozyme were delivered to rat aortic smooth muscle cells as described above. As in previous examples, percent inhibition was calculated by comparing the effects of the active ribozyme to the effects of the inactive ribozyme. As shown in FIG. 10, the ribozyme concentration at which cell proliferation is inhibited by 50% (IC.sub.50) is approximately 70 nM. From day to day, the IC.sub.50 varies between 25 and 100 nM.
Example 16
Direct comparison of the effects of ribozymes and antisense DNA.
Ribozymes are thought to be more specific reagents for the inhibition of gene expression than antisense oligonucleotides due to their catalytic activity and strict sequence requirements around the site of cleavage (Castanotto et al., 1994 Adv. in Pharmacol. 25, 289). To test this hypothesis, ribozyme activity was directly compared to the activity of phosphorothioate DNA oligonucleotides that target the same site in the c-myb mRNA. The ribozyme used was the 2'-C-allyl P=S-modified ribozyme described in Example 14, above. This ribozyme binds to a 15 nucleotide long region of the c-myb mRNA. Thus, a 15 nucleotide antisense phosphorothioate DNA molecule was prepared. A phosphorothioate DNA oligonucleotide with a randomly scrambled sequence of the same 15 nucleotides and a 2'-C-allyl P=S-modified ribozyme with randomly scrambled target binding arm sequences were synthesized as controls (by comparison to the murine c-myb cDNA sequence, the scrambled controls would not be expected to bind any region of the c-myb mRNA). Since longer phosphorothioate DNA oligonucleotides are often utilized as antisense inhibitors (for a review see Wagner, 1994 Science 372, 333), a symmetrically placed, 25 nucleotide phosphorothioate DNA antisense oligonucleotide and its scrambled sequence control were also synthesized. The ribozymes and the antisense oligonucleotides were delivered to rat smooth muscle cells as complexes with the cationic lipid, Lipofectamine, and serum-stimulated smooth muscle cell proliferation was measured subsequently.
As shown in FIG. 11, the 2'-C-allyl P=S-modified ribozyme demonstrated greater inhibition of smooth muscle cell proliferation than either of the antisense oligonucleotides. Furthermore, the scrambled arm ribozyme and inactive ribozyme controls demonstrated less non-specific inhibition than either of the scrambled sequence antisense control oligonucleotides. In fact, the non-specific inhibition demonstrated by the 25 nucleotide phosphorothioate molecule completely masked any specific effect of the antisense molecule. Similar results have been obtained with phosphorothioate DNA targeting other sites in the c-myb mRNA. Thus, a ribozyme that cleaves c-myb RNA is a more potent and more specific inhibitor of smooth muscle cell proliferation than phosphorothioate antisense DNA molecules.
Example 17
Chemically-modified ribozymes targeting different sites in the c-myb mRNA specifically inhibit smooth muscle cell proliferation.
If the observed inhibition of smooth muscle cell proliferation is mediated by ribozyme cleavage of c-myb mRNA, then other ribozymes that target the same mRNA should have the same effect. Two other ribozymes targeting two disparate sites in the c-myb mRNA (sites 549 and 1553, ribozyme Seq. ID Nos. 102 and 112) were synthesized with the 2'-C-allyl P=S modifications as described in Example 14. Inactive ribozyme controls also were synthesized corresponding to each new target sequence. Chemically-modified ribozymes targeting sites 549, 575, and 1553 were delivered to rat smooth muscle cells and their ability to inhibit serum-stimulated cell proliferation was assessed. Equivalent levels of inhibition are obtained with active ribozymes targeting sites 549, 575 and 1553 (see FIG. 12). None of the inactive ribozymes inhibited cell proliferation. Active ribozymes targeting other mRNA sequences not present in c-myb or ribozymes with scrambled arm sequences also fail to inhibit smooth muscle cell proliferation (see FIG. 12). Thus, inhibition of cell proliferation requires a catalytically active ribozyme that can bind to accessible c-myb mRNA sequences and is likely due to the reduction of c-myb mRNA levels by ribozyme cleavage.
Examples 18 and 19 describe experiments designed to determine the position and minimum number of phosphorothioate residues required for efficacy.
Example 18
Effect of position of phosphorothioate linkages on ribozyme inhibition.
Ribozymes targeting c-myb site 575 were synthesized with the 2'-C-allyl modification and with phosphorothioate linkages between various nucleotides in the ribozyme. One ribozyme contained a total of 10 phosphorothioate linkages, 5 in Stem I and 5 in Stem III, identical to the ribozyme described in Examples 14 through 17 above (referred to as 10 P=S 5' and 3' in FIG. 13A). One ribozyme contained only 5 phosphorothioate linkages in Stem III (5 P=S 3' in FIG. 13A). Another ribozyme contained 5 phosphorothioate linkages between the 6 nucleotides comprising the last base pair of stem 11 and the GAAA loop (5 P=S loop in FIG. 13A). The fourth ribozyme contained 5 phosphorothioate linkages in stem 1 (5 P=S 5' in FIG. 13A). The latter two ribozymes also were synthesized with the 3'--3' thymidine at the 3'end to help protect the ribozyme from 3' exonucleases (Ortigao et al., 1992 Antisense Res. & Development 2, 129; Seliger et al., Canadian Patent Application No. 2,106,819). The structure of these four different ribozymes is diagrammed in FIG. 13A. Inactive ribozyme controls were synthesized for each individual ribozyme. The active and inactive ribozymes were applied to rat smooth muscle cells as RNA/Lipofectamine complexes and their effects on cell proliferation were measured.
Referring to FIG. 13B, the ribozyme containing 5 phosphorothioate linkages in Stem I and the 3' inverted thymidine inhibited smooth muscle cell proliferation as well as the parent ribozyme with 10 total phosphorothioate linkages. None of the other ribozymes demonstrated significant differences between active and inactive controls. Therefore, the 3' inverted T can effectively substitute for the 5 phosphorothioate linkages in Stem III. Phosphorothioate linkages in the loop position lead to non-specific inhibition of smooth muscle cell proliferation, while phosphorothioate linkages in Stem I are necessary for enhanced efficacy in cells. Additionally, these results suggest that 3'-end modifications, such as iT, is desirable to minimize the amount of phosphorothioate contained in the ribozymes in order to minimize toxicity and facilitate chemical synthesis, while maintaining protection from endogenous 3'-exonuclease digestion.
Example 19
Minimizing phosphorothioate linkages in Stem I.
Fewer phosphorothioate linkages in the ribozyme will reduce the complexity and cost of chemical synthesis. Furthermore, phosphorothioate DNA molecules are known to have some undesirable and non-specific effects on cellular functions (for a review see Wagner, supra); reducing the phosphorothioate linkages in these RNA molecules is expected to enhance their specificity. A series of ribozymes targeting c-myb were synthesized to determine how many phosphorothioate linkages in Stem I are required for optimal ribozyme activity. The ribozymes contained 5, 4, 3, 2, or 1 phosphorothioate linkage(s) in Stem I, beginning with the phosphodiester bond between the first and second nucleotides and proceeding 3'. Each ribozyme contained the 2'-O-methyl modifications, the U.sub.4 2'-C-allyl nucleotide, and the inverted T nucleotide at the 3' end as described above. Activity of each of these ribozymes was compared to the activity of the ribozyme with 10 phosphorothioate linkages, 5 each in Stems I and III (referred to as 10 P=S in FIG. 14). Active and inactive ribozymes were applied to rat smooth muscle cells as complexes with Lipofectamine and their effects on smooth muscle cell proliferation were measured in two separate experiments. The results are diagrammed in FIG. 14. Ribozymes with 10, 5, and 4 phosphorothioate linkages showed equivalent efficacy. Ribozymes with fewer than four phosphorothioate linkages also showed efficacy, but the level of inhibition of smooth muscle cell proliferation was modestly reduced.
Example 20
Varying the length of Stems I and III
Ribozymes that cleave c-myb RNA at position 575 were synthesized with varying arm lengths. Each ribozyme contained 4 phosphorothioate linkages at the 5' end, 2'-O-methyl and 2'-C-allyl modifications and an inverted thymidine nucleotide at the 3'end as described above. FIG. 15 shows the effects of these ribozymes upon rat smooth muscle cell proliferation. Ribozymes were delivered at 100 nM with cationic lipid. Ribozymes with 6/6, 7/7 and 5/10 arms (where x/y denotes the nucleotides in Stem I/nucleotides in Stem III; see FIG. 2) all showed comparable efficacy. As shown in FIG. 15, ribozymes with longer arm lengths tended to demonstrate more non-specific inhibition (the inactive ribozyme controls with longer binding arms inhibited smooth muscle cell proliferation) when compared to ribozymes with shorter binding arms. From these data, it appears that ribozymes with 6/6, 7/7, 5/10, 10/5, 8/8 and 10/10 nucleotide arms all specifically inhibit smooth muscle cell proliferation, optimal inhibition, however, is observed with 6/6, 7/7 and 5/10 nucleotide arms.
Example 21
Ribozymes with different modified nucleotides inhibit smooth muscle cell proliferation.
Ribozymes containing seven nucleotides in both Stems I and II, four phosphorothioate residues at the 5' end and a 3'--3' inverted thymidine at the 3' end, were synthesized with various modified nucleotides at the U.sub.4 and U.sub.7 positions within the core of a HH ribozyme. All of the modified catalytic core chemistries retained ribozyme activity and demonstrated enhanced stability to serum nucleases (Usman et al., 1994 supra). The ribozyme termed U4 2'-C-allyl contains a 2'-C-allyl uridine at the U.sub.4 position and a 2'-O-methyl nucleotide at the U.sub.7 position. The ribozyme termed U4,U7 2'-amino contains a 2'-amino nucleotide at both U4 and U7. The ribozyme termed U4 2'-fluoro contains a 2'-fluoro-modified nucleotide at U4 and 2'-O-methyl at U7. The ribozyme termed U4 6-methyl contains a 6-methyl uridine nucleotide at U4 and 2'-O-methyl at U7. The ribozyme termed U4 deoxyabasic contains a deoxyribose moeity and lacks a base at U4 (Beigelman et al., 1994 Bioorganic & Med. Chem. Letters 4, 1715) and 2'-O-methyl at U7. Active and inactive versions of each of the chemically-modified ribozymes were applied to rat smooth muscle cells using Lipofectamine as described above. As diagrammed in FIG. 16, all of the nuclease-stable, chemically-modified ribozymes demonstrated significant inhibition of rat smooth muscle cell proliferation. Thus, the requirements for ribozyme activity in smooth muscle cells appear to be a catalytically core that is modified to minimize endonucleolytic degradation and modifications at the 5' and 3' ends which may prevent exonucleolytic degradation.
Chemical modifications described in this invention are meant to be non-limiting examples, and those skilled in the art will recognize that other modifications (base, sugar and phosphate modifications) to enhance nuclease stability of a ribozyme can be readily generated using standard techniques and are hence within the scope of this invention.
Example 22
Ribozyme inhibition of pig smooth muscle cell proliferation.
Of the commonly used animal models of intimal hyperplasia after balloon angioplasty, the pig model is believed to be most predictive of human disease (Steele et al., 1985 Circ. Res. 57, 105; Ohno et al., 1994 Science 265, 781; Baringa, 1994 Science 265, 738). Therefore, we wished to assess the ability of c-myb ribozymes to inhibit pig smooth muscle cell proliferation. Yucatan pig smooth muscle cells (YSM) were obtained from Dr. Elizabeth Nabel (University of Michigan Medical Center) and were grown in Dulbecco's modified Eagle's medium as described (see Example 6). The YSM cells were starved for 72 hours in DMEM with 0.1% FBS. Active and inactive ribozymes (four phosphorothioate linkages at the 5' end, 2'-C-allyl-modified core and 3'--3' inverted thymidine at the 3' end) were applied as RNA/Lipofectamine.RTM. complexes as described in the above examples. Proliferation was stimulated with serum and assessed by BrdU incorporation. FIG. 17 shows that a ribozyme dose of as low as 75 nM can inhibit pig smooth muscle cell proliferation by as much as 60%. The same chemical modifications of the ribozymes (2'-modified, stable core, 5' phosphorothioate linkages and 3' inverted thymidine) are required to obtain significant and reproducible inhibition of pig smooth muscle cell proliferation as were shown to be required for inhibition of rat cells in the above Examples.
Example 23
Ribozyme inhibition of human smooth muscle cell proliferation.
In Example 12, we demonstrated that a minimally modified ribozyme directed against c-myb site 549 could significantly inhibit human smooth muscle cell proliferation. The 2'-C-allyl and phosphorothioate-modified ribozyme targeting c-myb site 575 characterized above was applied to human smooth muscle cells as RNA/Lipofectamine.RTM. complexes. Inactive ribozyme and inactive, scrambled arm ribozymes were applied as controls. At 200 nM, the active ribozyme inhibits human smooth muscle proliferation by greater than 75% while the inactive ribozyme inhibits proliferation by only 38%. The ribozyme with scrambled binding arm sequences fails to inhibit. At 100 nM, the active ribozyme still demonstrates significant inhibition while neither the inactive or scramble controls inhibit cell proliferation (see FIG. 18). Thus, the active ribozyme identified in these studies mediates significant inhibition of human smooth muscle cell proliferation and represents a novel therapeutic for restenosis and/or vascular disease.
Example 24
Delivery of c-myb ribozymes to vessels in vivo.
The ribozyme that cleaves c-myb RNA at site 575 was synthesized in two parts (Mamone, supra), the internal 5' end was labeled with .sup.33 P using polynucleotide kinase and the two fragments were ligated with RNA ligase. The resulting RNA was an intact ribozyme with an internal .sup.33 P label. This internally-labeled ribozyme was delivered to balloon injured rat carotid arteries as described (Simons et al., 1992 Nature 359, 67). Rats were anesthetized and the carotid artery was surgically exposed. The external carotid was dissected and a 2F Fogarty balloon catheter was inserted and directed into the carotid artery. Injury was caused by repeated (3 times) inflation and retraction of the balloon. The injured region was isolated by ligatures and a cannula was inserted in the external carotid. Ribozymes alone (two rat vessels) or ribozyme/Lipofectamine.RTM. complexes (two rat vessels) were applied to the injured vessel through the cannula and were left in the vessel for twenty minutes. After application, blood flow was restored by removal of the ligatures for five minutes and the vessels were harvested and processed as described below.
Half of the vessel was frozen in liquid nitrogen, crushed into a fine powder, and RNA was extracted using standard protocols. The extracted RNA was applied to a denaturing polyacrylamide gels and subjected to electrophoresis. Autoradiography of the gel permitted detection of the .sup.33 P label; the amount of radioactivity in each band was quantitated using a Phosphor-imaging system. The amount of extracted and intact ribozyme was calculated by direct comparison to labeled ribozyme controls run on the same gel. The percentage of the ribozyme delivered intact could be estimated by quantifying the percentage of label that co-migrates with the intact ribozyme controls. After delivery of ribozymes in phosphate-buffered saline (PBS), 3% of the .sup.33 P label was recovered from the rat vessels and >90% of the label was present in the form of intact ribozyme. After delivery of ribozyme in RNA/Lipofectamine complexes, 10 to 11% of the .sup.33 P label was recovered from the rat vessels and 20 to 90% of the label was present in the form of intact ribozyme. The significant uptake of the intact ribozyme demonstrates that local delivery of modified ribozymes to arterial walls is feasible.
The other half of each vessel was fixed in PBS-buffered 2% glutaraldehyde, sectioned onto slides and coated with emulsion. After autoradiography for four days, the emulsion was developed and the sections were stained with hematoxylin and eosin by standard techniques (Simons et al., 1992 supra). Inspection of the sections showed a majority of the grains present over the medial smooth muscle cells after application of the ribozyme. Some .sup.33 P label could be detected in the underlying adventitia as well. Similar density and distribution of grains was observed when the ribozyme was delivered with or without Lipofectamine. These data demonstrate that ribozyme can penetrate the injured vessel wall and is in close apposition or within the underlying medial smooth muscle cells. Thus, therapeutic ribozymes can be locally delivered to vessels for the treatment of vascular disease.
Similar experiments were performed in pig iliofemoral vessels. After balloon injury, a ribozyme, internally labeled with .sup.33 P as described above, was delivered with a double balloon catheter device (Nabel and Nabel, supra; Ohno et al., 1994 supra). After 20 minutes, blood flow was restored by deflating the balloons. The vessels were harvested after an additional hour or the surgical injuries were sutured and the vessels harvested one day later. Harvested vessels were sectioned, subjected to autoradiography and stained. One hour after delivery, the majority of the .sup.33 P label could be detected in the media, overlying or within smooth muscle cells. Some label was also detected at the luminal surface of the vessel and in the adventitial tissue. One day after delivery, grains could be still be detected associated with remaining medial smooth muscle cells. No major differences in density or distribution was observed between ribozymes delivered with or without Lipofectamine.RTM.. These data demonstrate that ribozymes can be locally delivered to smooth muscle cells of injured vessels in a large animal model that is clinically relevant to human vascular disease.
Example 25
Ribozyme-mediated decrease in the level of c-myb RNA in rat smooth muscle cells.
To determine whether a ribozyme catalyzes the cleavage of c-myb RNA in a mammalian cell, applicant has used a sensitive quantitative competitive polymerase chain reaction (QCPCR) to assay the level of c-myb RNA in rat smooth muscle cells treated with either catalytically active or inactive ribozyme.
Rat smooth muscle cells (RASMC) were treated with ribozymes as described above. Following the ribozyme treatment for 4 h, cells were stimulated with 10% serum (in the presence or absence of BrdU). After 24 h, cells were harvested for further analysis. Cells, that were treated with BrdU, were assayed for proliferation as described above. Cells, that were not treated with BrdU, were used for the QCPCR assay.
The following is a brief description of the QCPCR technique used to quantitate levels of c-myb mRNA from RASMC, normalizing to the housekeeping gene, GAPDH. This method was adapted from Thompson et al, Blood 79:1692, 1992. Briefly, total RNA was isolated from RASMC using the Guanidinium isothiocyanate technique of Chomczynski and Sacchi (Analytical Biochemistry, 162:156, 1987). In order to construct a deletion competitor and control wild-type RNA, a cDNA clone of the rat c-myb message, referred to as pc8myb, was used. The competitor RNA comprises a deletion of 50 bases, making it smaller than the wild-type cellular RNA, and spansfrom nucleotide 428 to nucleotide 753.
A house-keeping gene, GAPDH, that is constitutively expressed by the RASMC, was used as an internal control for QCPCR assay. A deletion competitor and wild-type controls for GAPDH were made the same way as for c-myb. GAPDH-containing plasmid (pTri-GAPDH) was purchased from Ambion. The GAPDH competitor is also a deletion mutant, lacking 50 bases. The GAPDH competitor was used to quantitate the amount of this housekeeping gene in each sample, thus allowing for a confirmation of cellular RNA's integrity and for the efficiency of RNA isolation. All quantitations for the level of c-myb expression were normalized to the level of GAPDH expression in the same sample of cells.
Referring to FIG. 19, RASMC that were treated with a stabilized catalytically active 575 HH ribozyme did not proliferate well. There was greater than 70% inhibition of RASMC proliferation when compared with approximately 25% inhibition of cell proliferation by a catalytically inactive version of the 575 HH ribozyme. The level of inhibition of RASMC proliferation correlates very well with the greater than 70% decrease in the level of c-myb RNA. This shows that the inhibition of smooth muscle cell proliferation is directly mediated by the cleavage of c-myb RNA by a ribozyme in RASMC.
FIG. 20 shows what Applicant presently believes is an optimal ribozyme configuration.
Example 26
Inhibition of smooth muscle cell proliferation by 2-5A antisense chimera.
By "2-5A antisense chimera" is meant, an antisense oligonucleotide containing a 5' phosphorylated 2'-5'-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which in turn cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
RNAs containing 2'-5' Adenosine with a terminal 5' phosphate has been shown to activate RNAse L (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300). The terminal phosphate is required for efficient activation of RNAse L. Ribozymes targeting c-myb site 575 were synthesized with 2-5A moieties on the 5' end, with and without the terminal 5' phosphate. The ribozyme-2-5A chimera was complexed with LipofectAMINE and assayed on rat aortic smooth muscle cells (RASMC) as described above.
As shown in FIG. 21, when no terminal phosphate is present, the active ribozyme 575 active Rz+inactive (A)4! functions similarly to a normal active ribozyme lacking a 2-5A modification (575 active Rz). An inactive ribozyme core with 5' phosphate-2-5A 575 inactive Rz+active P(A)4! shows significant inhibition relative to the controls, but has significantly lower activity when compared with an active ribozyme. A molecule that contains both an active ribozyme core and 5' phosphate-contining 2-5A 575 active Rz+active P (A)4! shows even greater inhibition than that obtained by either mechanism individually, inhibiting the smooth muscle cell proliferation to baseline levels (0% FBS). Thus the ribozyme and 2-5A anitisense chimera together show an additive effect in inhibiting RASMC proliferation.
Use of Ribozymes That Cleave c-myb RNA to Treat Restenosis.
The above discussion demonstrates, by way of example, how ribozymes that inhibit smooth muscle cell proliferation are delivered directly, or through the use of expression vectors, to vessels. Preferably, ribozymes cleaving c-myb RNA are delivered to vessels at the time of coronary angioplasty. Local delivery during intervention can be achieved through the use of double balloon catheters , porous balloon catheters, balloon catheters coated with polymers (Riessen, R., et al., 1993, Human Gene Therapy, 4, 749-758), or biopolymer stents (Slepian and Schindler, U.S. Pat. No. 5,213,580). In the above examples, ribozymes were identified that could inhibit roughly half of the smooth muscle cells in culture from proliferating in response to the growth factors present in serum. A corresponding 50% (or even lower) reduction in intimal thickening will significantly improve the outcome of patients undergoing coronary angioplasty.
Use of Ribozymes Targeting c-myb to Treat Cancer
Overexpression of the c-myb oncogene has been reported in a number of cancers, including leukemias, neuroblastomas, and lung, colon, and breast carcinomas (Torelli, G., et al., 1987, Cancer Res., 47, 5266-5269; Slamon, D. J., et al., 1986, Science, 233, 203-206; Slamon, D. J., et al., 1984, Science, 224, 256-262; Thiele, C. J., et al., 1988, Mol. Cell. Biol., 8, 1677-1683; Griffin, C. A. and Baylin, S. B., 1985, Cancer Res., 45, 272-275; Alitalo, K., et al., 1984, Proc, Natl. Acad. Sci. USA, 81, 4534-4538). Thus, inhibition of c-myb expression can reduce cell proliferation of a number of cancers. Indeed, in tissue culture, treatment of colon adenocarcinoma, neurectodermal, and myeloid leukemia cell lines with antisense c-myb oligonucleotides inhibits their proliferation (Melani, C., et al., 1991, Cancer Res., 51, 2897-2901; Raschella, F., et al., 1992, Cancer Res., 52, 4221-4226; Anfossi, G., et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 3379-3383). Furthermore, myeloid cells from patients with chronic myelogenous leukemia and acute myelogenous leukemia are differentially sensitive to c-myb antisense oligonucleotides (Calabretta, B., et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 2351-2355). Ratajczak, et al. (1992, Proc. Natl. Acad, Sci. USA, 89, 11823-11827) treated mice bearing human leukemia cells with c-myb antisense oligonucleotides and significantly prolonged their survival and reduced their tumor burden. Thus, reduction of c-myb expression in leukemic cells in tissue culture and in vivo can reduce their proliferative potential.
While the above studies demonstrated that antisense oligonucleotides can efficiently reduce the expression of c-myb in cancer cells and reduce their ability to proliferate and spread, this invention describes the first enzymatic RNAs, or ribozymes, shown to cleave c-myb RNA. Such ribozymes, with their catalytic activity and increased site specificity (see above), are likely to represent more potent and safe therapeutic molecules than antisense oligonucleotides for the treatment of cancer as well as restenosis. In the present invention, ribozymes are shown to inhibit smooth muscle cell proliferation. From those practiced in the art, it is clear from the examples described, that the same ribozymes may be delivered in a similar fashion to cancer cells to block their proliferation.
In a preferred embodiment, autologous bone marrow from patients suffering with acute myelogenous leukemia or chronic myelogenous leukemia are treated with ribozymes that cleave c-myb RNA. Ribozymes will be delivered to the autologous bone marrow cells ex vivo at 0.1 to 50 .mu.M with or without forming complexes of the ribozymes with cationic lipids, encapsulating in liposomes or alternative delivery agents. After several days, the proliferative capacity of the leukemic cells in the patients bone marrow will be reduced. The patient's endogenous bone marrow cells will be depleted by chemical or radiation treatments and their bone marrow reconstituted with the ex vivo treated cells. In such autologous bone marrow reconstitution treatments of leukemic patients, recurrence of the disease can be caused by proliferation of leukemic cells present in the transplanted bone marrow. Significantly reducing the proliferative potential of the leukemic cells by treating with ribozymes that cleave c-myb RNA will reduce the risk of recurrent leukemia.
Diagnostic uses
Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of c-myb RNA in a cell. 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 vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNAs associated with c-myb related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., c-myb) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
Other embodiments are within the following claims.
Claims
Certificate of Correction present
The claims shown below may not include correction changes. Use the corrected PDF for authoritative text.
We claim:
1. An enzymatic RNA molecule for the specific cleavage of c-myb RNA, wherein said enzymatic RNA molecule comprises an oligonucleotide having a 2'-5'-linked adenylate residue having a 5'-phosphate.
2. The enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule is complementarity to c-myb RNA at a region selected from the group consisting of: SEQ ID Nos. 1-100, 130-148, and even numbered SEQ ID Nos. from SEQ ID No. 170 to 1224.
3. The enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule is complementarity to c-myb RNA at a region other than the region consisting of nucleotides 111-140.
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