Definitions

• this invention relates to methods for inhibition of osteoarthritis, in particular, inhibition of genetic expression which leads to a reduction or elimination of extracellular matrix digestion by matrix metalloproteinases.
• osteoarthritis is a slowly progressive disease characterized by degeneration of articular cartilage with proliferation and remodeling of subchondral bone. It presents with a clinical picture of pain, deformity, and loss of joint motion.
• Rheumatoid arthritis is a chronic systemic inflammatory disease. Rheumatoid arthritis may be mild and relapsing or severe and progressive, leading to joint deformity and incapacitation.
• Arthritis is the major contributor to functional impairment among the older population. It is the major cause of disability and accounts for a large proportion of the hospitalizations and health care expenditures of the elderly. Arthritis is estimated to be the principal cause of total incapacitation for about one million persons aged 55 and older and is thought to be an important contributing cause for about one million more.
• osteoarthritis is diagnosed objectively on the basis of reading radiographs, but many people with radiologic evidence of disease have no obvious symptoms.
• rheumatoid arthritis has a world-wide distribution and affects all racial and ethnic groups. The exact prevalence in the US is unknown but has been estimated to range between 0.5% and 1.5%. Rheumatoid arthritis occurs at all age levels and generally increases in prevalence with advancing age. It is 2-3 times more prevalent in women than in men and peak incidence occurs between 40-60 years of age. In addition to immunological factors, environmental, occupational and psychosocial factors have been studied for potential etiologic roles in the disease.
• the extracellular matrix of multicellular organisms plays an important role in the formation and maintenance of tissues.
• the meshwork of the extracellular matrix is deposited by resident cells and provides a framework for cell adhesion and migration, as well as a permeability barrier in cell-cell communication.
• Connective tissue turnover during normal growth and development or under pathological conditions is thought to be mediated by a family of neutral metalloproteinases, which are zinc-containing enzymes that require calcium for full activity.
• the regulation of metalloproteinase expression is cell-type specific and may vary among species.
• interstitial collagenase The best characterized of the matrix metalloproteinases, interstitial collagenase (MMP-1), is specific for collagen types I, II, and III. MMP-1 cleaves all three chains of the triple helix at a single point initiating sequential breakdown of the interstitial collagens. Interstitial collagenase activity has been observed in rheumatoid synovial cells as well as in the synovial fluid of patients with inflammatory arthritis.
• Gelatinases (MMP-2) represent a subgroup of the metalloproteinases consisting of two distinct gene products; a 70 kDa gelatinase expressed by most connective tissue cells, and a 92 kDa gelatinase expressed by inflammatory phagocytes and tumor cells.
• the larger enzyme is expressed by macrophages, SV-40 transformed fibroblasts, and neutrophils.
• the smaller enzyme is secreted by H-ras transformed bronchial epithelial cells and tumor cells, as well as normal human skin fibroblasts. These enzymes degrade gelatin (denatured collagen) as well as native collagen type XI.
• Stromelysin (MMP-3) has a wide spectrum of action on molecules composing the extracellular matrix. It digests proteoglycans, fibronectin, laminin, type IV and IX collagens and gelatin, and can remove the N-terminal propeptide region from procollagen, thus activating the collagenase. It has been found in human cartilage extracts, rheumatoid synovial cells, and in the synovium and chondrocytes of joints in rats with collagen-induced arthritis.
• tissue growth factor- ⁇ has been shown to block epidermal growth factor (EGF) induction of stromelysin synthesis in vitro.
• Experimental protocols involving gene therapy approaches include the controlled expression of the metalloproteinase inhibitors TIMP-1 and TIMP-2. Of the latter three approaches, only ⁇ -interferon treatment is currently feasible in a clinical application.
• the invention relates to methods for the induction of graft tolerance, treatment of autoimmune diseases, inflammatory disorders and allergies in particular, by inhibition of B7-1 , B7-2, B7-3 and CD40.
• T lymphocytes T cells
• T cell activation is a multi-step process requiring several signalling events between the T cell and an antigen presenting cell.
• the ensuing discussion details signals that are exchanged between T cells and antigen presenting B cells. Similar pathways are thought to occur between T cells and other antigen presenting cells such as monocytes or follicular dendritic cells.
• T cell activation is initiated when the T-cell receptor (TCR) binds to a specific antigen that is associated with the MHC proteins on the surface of an antigen presenting cell.
• TCR T-cell receptor
• This primary stimulus activates the T cell and induces expression of CD40 ligand (CD40L) on the surface of the T cell.
• CD40L then interacts with its cognate receptor, CD40, which is constitutively expressed on the surface of B cells; CD40 transduces the signal leading to B cell activation.
• B cell activations result in the expression of B7-1 , B7-2 and/or B7-3, whiGh in turn interacts with constitutively expressed CD28 on the surface of T cells. The interaction generates a secondary co-stimulatory signal that is required to fully activate the T cell.
• T cell activation via the T cell receptor and CD28 leads to cytokine secretion, clonal expansion, and differentiation. If the T cell receptor is engaged, absence of this secondary co-stimulus mediated by CD28, then the T cell is inactivated, either by clonal anergy (non- responsiveness or reduced reactivity of the immune system to specific antigen(s)) or clonal deletion (Jenkins et al., 1987 Proc. NatI. Acad. Sci. USA 84, 5409). Thus, engagement of the TCR without a concommitant costimulatory signal results in a state of tolerance toward the specific antigen recognized by the T cell.
• This co-stimulatory signal can be mediated by the binding of B7-1 or B7-2 or B7-3, present on activated antigen-presenting cells, to CD28, a receptor that is constitutively expressed on the surface of the T cell (Marshall et al., 1993 J Clin Immun 13, 165-174; Linsley, et al., 1991 J Exp Med 173, 721 ; Koulova et al., 1991 J Exp Med 173, 759; Harding et al., 1992 Nature 356, 607).
• B7-1 and B7-3 are only expressed on the surface of a subset of B cells after 48 hours of contact with T cells.
• B7-2 mRNA is constitutively expressed by unstimulated B cells and increases 4-fold within 4 hours of activation (Freeman et al., 1993Sc7ence 262, 909; Boussiotis et al., 1993 Proc NatI Acad Sci USA 90, 11059). Since T cells commit to either the anergy or the activation pathway within 12-24 hours of the initial TCR signal, it is thought that B7-2 is the molecule responsible for the primary costimulatory signal. B7-1 and B7-3 may provide a subsequent signal necessary for clonal expansion.
• Antibodies to B7-2 completely block T cell proliferation in a mixed lymphocyte reaction (Azuma et al., 1993 supra), supporting the central role of B7-2 in T cell activation.
• B7 (B7-1 ) is a 60 KD modified trans-membrane glycoprotein usually present on the surface of antigen presenting cells (APC). B7 has two ligands- CD28 and CTLA4. Interaction of B7-1 with CD28 and/or CTLA4 causes activation of T cell responses (Janeway and Bottomly, 1994 Cell 76, 275).
• B7-2 is a 70 KD (34 KD unmodified) trans-membrane glycoprotein found on the surface of APCs. B7-2 encodes a 323 amino-acid protein which is 26
• B7-1 % identical to human B7-1 protein.
• CD28 and CTLA4 are selectively bound by B7-2.
• B7-2 unlike B7-1 , is expressed on the surface of unstimulated B cells (Freeman et al., 1993 supra).
• CD40 is a 45-50 KD surface glycoprotein found on the surface of late pre-B cells in bone marrow, mature B cells, bone marrow-derived dendritic cells and follicular dendritic cells (Clark and Ledbetter, 1994 Nature 367, 425).
• organ graft rejection is mediated by T cell effector function
• the goal is to block specifically the activation of the subset of T cells that recognize donor antigens.
• a limitation in the field of transplantation is the supply of donor organs (Nowak 1994 Science 266, 1 148).
• the ability to induce donor-specific tolerance would substantially increase the chances of successful allographs, xenographs, thereby greatly increasing the donor pool.
• transplantation includes grafting of tissues and/or organ ie., implantation or transplantation of tissue and/or organs, from the body of an individual to a different place within the same or different individual. Transplantation also involve grafting of tissues and/or organs from one area of the body to another. Transplantation of tissues and/or organs between genetically dissimilar animals of the same species is termed as allogeneic transplantation. Transplantation of animal organs into humans is termed xenotransplants (for a review see Nowak, 1994 Science 266, 1148).
• CTLA4-lg fusion protein is a homologue of CD28 that binds B7-1 and B7-2 with high affinity.
• the engineered, soluble fusion protein, CTLA4-lg binds B7-1 , thereby blocking its interaction with CD28.
• the results of CTLA4-lg treatment in animal studies are mixed.
• CTLA4-lg treatment significantly enhanced survival rates and ameliorated the symptoms of graft-versus host disease in a murine bone marrow tranplant model (Blazer et al., 1994 Blood 83, 3815).
• CTLA4-lg induced long-term (>110 days) donor-specific tolerance in pancreatic islet xenographs (Lenschow et al., 1992Science 257, 789). Conversely, in another study CTLA4-lg treatment delayed but did not ultimately prevent cardiac allograft rejection (Turka, et al., 1992 Proc NatI Acad Sci U S A 89, 11102). Mice immunized with sheep erythrocytes in the presence of CTLA4-lg failed to mount a primary immune response (Linsley, et al., 1992 Science 257, 792). A secondary immunization did elicit some response, however, indicating incomplete tolerance.
• CTLA4-lg was administered 2 days after primary immunization, leading the authors to conclude that CTLA4-lg blocked amplification rather than initiation of the immune response. Since CTLA4-lg has been shown to dissociate more rapidly from B7-2 compared with B7-1 , this may explain the failure to induce long term tolerance in this model (Linsley et al., 1994 Immunity ! , 793).
• CTLA4:lg has recently been shown to ameliorate symptoms of spontaneous autoimmune disease in lupus-prone mice (Finck et al., 1994 Science 265, 1225).
• the B7 antigen, or its fragments or derivatives are reacted with CD28 positive T cells to regulate T cell interactions with other cells B7 antigen or CD28 receptor may be used to inhibit interaction of cells associated with these molecules, thereby regulating T cell responses.”
• WO 94/01547 describe the use of anti-B7 and anti- CD40 antibodies to treat allograft transplant rejection, graft versus host disease and rhematoid arthritis.
• the application states that- "...anti-B7 and anti-CD40 antibodies...can be used to prevent or treat an antibody- mediated or immune system disease in a patient.”
• Therapeutic agents used to prevent rejection of a transplanted organ are all cytotoxic compounds or antibodies designed to suppress the cell-mediated immune system.
• the side effects of these agents are those of immunosuppression and infections.
• the primary approved agents are azathioprine, corticosteroids, cyclosporine; the antibodies are antilymphocyte or antithymocyte globulins. All of these are given to individuals who have been as closely matched as possible to their donors by both major and minor histocompatibility typing. Since the principal problem in transplantation is an antigenic mismatch and the resulting need for cytotoxic therapy, any therapeutic improvement which decreases the local immune response without general immunosuppression should capture the transplant market.
• Cyclosporine At the end of the 1970's and early 1980's the introduction of cyclosporine revolutionized the transplantation field. It is a potent immunosuppressant which can inhibit immunocompetent lymphocytes specifically and reversibly. Its primary mechanism of action appears to be inhibition of the production and release of interleukin-2 by T helper cells. In addition it also interferes with the release of interleukin-1 by macrophages, as well as proliferation of B lymphocytes. It was approved by the FDA in 1983 and by 1989 was almost universally given to transplant recipients.
• Azathioprine In addition to cyclosporine, azathioprine is used for transplant patients. Azathioprine is one of the mercaptopurine class of drugs and inhibits nucleic acid synthesis. Patients are maintained indefinitely on daily doses of 1 mg/kg or less, with a dosage adjusted in accordance with the white cell count. The drug may cause depression of bone marrow elements and may cause jaundice.
• Corticosteroids Prednisone, used in almost all transplant recipients, is usually given in association with azathioprine and cyclosporine. The dosage must be regulated carefully so as so prevent complications such as infection, development of cushingoid features, and hypertension. Usually the initial maintenance prednisone dosage is 0.5 mg/kg/d. This dosage is usually further decreased in the outpatient clinic until maintenance levels of about 10 mg/d for adults are obtained. The exact site of action of corticosteroids on the immune response is not known.
• Tacrolimus On April 13, 1994 the Food and Drug Administration approved another drug to help prevent the rejection of organ transplants. The drug, tacrolimus, was approved only for use in liver transplant patients.
• An alternative to cyclosporine, the macrolide immunosuppressant tacrolimus is a powerful and selective anti-T-lymphocyte agent that was discovered in 1984.
• Tacrolimus isolated from the fungus Streptomyces tsukubaensis, possesses immunodepressant properties similar to but more potent than cyclosporine. It inhibits both cell-mediated and humoral immune responses. Like cyclosporine, tacrolimus demonstrates considerable interindividual variation in its pharmacokinetic profile.
• tacrolimus has shown notable efficacy as a rescue or primary immunosuppressant therapy when combined with corticosteroids.
• the potential for reductional withdrawal of corticosteroid therapy with tacrolimus appears to be a distinct advantage compared with the cyclosporine. This benefit may be enhanced by reduced incidence of infectious complications, hypertension and hypercholesterolemia reported by some investigators.
• the tolerability profile of tacrolimus appears to be broadly similar to that of cyclosporine.
• T cell anergy can be used to reverse autoimmune diseases.
• Autoimmune diseases represent a broad category of conditions.
• a few examples include insulin-dependent diabetes mellitus (IDDM), multiple schlerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), myasthenia gravis (MG), and psoriasis.
• IDDM insulin-dependent diabetes mellitus
• MS multiple schlerosis
• SLE systemic lupus erythematosus
• RA rheumatoid arthritis
• MG myasthenia gravis
• psoriasis psoriasis.
• Chappel, WO 94/11011 describes methods to treat autoimmune diseases by inducing tolerance to cells, tissues and organs.
• the application states that- "Cells genetically engineered with DNA encoding a plurality of antigens of a cell, tissue, or organ to which tolerance is to be induced.
• the cells are free of co-stimulatory antigens, such as B7 antigen.
• Such cells induce T-cell anergy against the proteins encoded by the DNA, and may be administered to a patient in order to prevent the onset of or to treat an autoimmune disease, or to induce tolerance to a tissue or organ prior to transplantation.”
• Allergic reactions represent an immediate hypersensitivity response to environmental antigens, typically mediated by IgE antibodies.
• the ability to induce antigen-specific tolerance provides a powerful avenue to alleviate allergies by exposure to the antigen in conjunction with down-regulation of B7-1 , B7-2, B7-3 or CD40.
• B7-1 , B7-2 and B7-3 in T cell activation remains to be determined. Some studies suggest that their functions are essentially redundant (Hathcock et al 1994 J Exp. Med. 180, 631), or that the differences observed in the kinetics of expression might simply indicate that B7-2 is important in the initiation of the co-stimulatory signal, while B7-1 plays a role in the amplification of that signal. Other studies point to more specific functions. For example, Kuchroo et al., 1995 Cell 80, 707, have reported that blocking B7-1 expression may favor a Th2 response, while blocking B7-2 expression favors a Th1 response. These two helper T cell subpopulations play distinct roles in the immune response and inflammatory disease. Th1 cells are strongly correlated with auto-immune disease. Allergic responses are typically triggered by Th2 response. Therefore, the decision to target B7-1 , B7-2, CD40 or a combination of the above will depend to the particular disease application.
• Ribozyme treatment can be a partner to current treatments which primarily target immune cells reacting to pre-existing tissue damage. Early ribozyme or antisense treatment which reduces the collagenase or stromelysin-induced damage can be followed by treatment with the anti-inflammatories or retinoids, if necessary. In this manner, expression of the proteinases can be controlled at both transcriptional and translational levels. Ribozyme or antisense treatment can be given to patients expressing radiological signs of osteoarthritis prior to the expression of clinical symptoms.
• Ribozyme or antisense treatment can impact the expression of stromelysin without introducing the non-specific effects upon gene expression which accompany treatment with the retinoids and dexamethasone.
• the ability of stromelysin to activate procollagenase indicates that a ribozyme or antisense molecule which reduces stromelysin expression can also be used in the treatment of both osteoarthritis (which is primarily a stromelysin- associated pathology) and rheumatoid arthritis (which is primarily related to enhanced collagenase activity).
• cytokines and growth factors induce metalloproteinase activities during wound healing and tissue injury of a pre- osteoarthritic condition
• these molecules are not preferred targets for therapeutic intervention.
• Primary emphasis is placed upon inhibiting the molecules which are responsible for the disruption of the extracellular matrix, because most people will be presenting radiologic or clinical symptoms prior to treatment.
• the most versatile of the metalloproteinases (the molecule which can do the most structural damage to the extracellular matrix, if not regulated) is stromelysin. Additionally, this molecule can activate procollagenase, which in turn causes further damage to the collagen backbone of the extracellular matrix.
• TIMPs tissue inhibitors of MMP
• the invention features use of specific ribozyme molecules to treat or prevent arthritis, particularly osteoarthritis, by inhibiting the synthesis of the prostromelysin molecule in synovial cells, or by inhibition of other matrix metalloproteinases discussed above.
• Cleavage of targeted mRNAs stromelysin mRNAs, including stromelysin 1 , 2, and 3, and collagenase expressed in macrophages, neutrophils and synovial cells represses the synthesis of the zymogen form of stromelysin, prostromelysin.
• Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. It is said that such enzymatic RNA molecules can be targeted to virtually any RNA transcript and efficient cleavage has been achieved in vitro. Kim et al., 84 Proc. Nat. Acad. of Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acid Research 1371 , 1989.
• 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.
• 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.
• enzymatic RNA molecule an RNA molecule which has complementarity in a substrate binding region to a specified mRNA target, and also has an enzymatic activity which is active to specifically cleave that mRNA. That is, the enzymatic RNA molecule is able to intermolecularly cleave mRNA and thereby inactivate a target mRNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur.
• One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. For in vivo treatment, complementarity between 30 and 45 bases is preferred; although lower numbers are also useful.
• nucleotide sequence that can form hydrogen bond(s) with other nucleotide sequence by either traditional Watson-Crick or other non-traditional types (for example Hoogsteen type) of base-paired interactions.
• ribozyme The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the 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.
• a single ribozyme molecule is able to cleave many molecules of target RNA.
• the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
• 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.
• hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampel et al., EPA 0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al., 1990 Nucleic Acids Res.
• 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 stromelysin encoding mRNAs 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.
• the ribozymes can be expressed from DNA or RNA vectors that are delivered to specific cells.
• nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
• small enzymatic nucleic acid motifs e.g., of the hammerhead or the hairpin structure
• the simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure.
• these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters (e.g., Scanlon et al., 1991 , Proc. NatI. Acad. Sci. USA. 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res.
• ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992 Nucleic Acids Svmp. 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) .
• Ribozyme therapy due to its extraordinar specificity, is particularly well- suited to target mRNA encoding factors that contribute to disease pathology.
• ribozymes that cleave stromelysin mRNAs may represent novel therapeutics for the treatment of asthma.
• the invention features ribozymes that inhibit stromelysin production.
• 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 stromelysin encoding mRNAs, preventing translation and stromelysin protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.
• inhibitor is meant that the activity or level of stromelysin encoding mRNAs and protein is reduced below that observed in the absence of the ribozyme, 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.
• 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 stromelysin activity in a cell or tissue.
• related is meant that the inhibition of stromelysin mRNAs and thus reduction in the level of stromelysin 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 RNA or RNA complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, aerosol inhalation, infusion pump or stent, with or without their incorporation in biopolymers.
• the ribozymes have binding arms which are complementary to the sequences in Tables All, AMI, AIV, AVI, AVIII and AIX. Examples of such ribozymes are shown in Tables AV, AVII, AVIII and AIX. Examples of such ribozymes consist essentially of sequences defined in these Tables.
• the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
• the invention features ribozymes that cleave target molecules and inhibit stromelysin 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.
• the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells.
• viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary.
• 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.
• vectors is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
• This class of chemicals exhibits a high degree of specificity for cleavage of the intended target mRNA. Consequently, the ribozyme agent will only affect cells expressing that particular gene, and will not be toxic to normal tissues.
• the invention can be used to treat or prevent (prophylactically) osteoarthritis or other pathological conditions which are mediated by metalloproteinase activation.
• the preferred administration protocol is in vivo administration to reduce the level of stromelysin activity.
• the invention features an enzymatic RNA molecule (or ribozyme) which cleaves mRNA associated with development or maintenance of an arthritic condition, e.g.. mRNA encoding stromelysin, and in particular, those mRNA targets disclosed in the accompanying tables, which include both hammerhead and hairpin target sites.
• an enzymatic RNA molecule or ribozyme which cleaves mRNA associated with development or maintenance of an arthritic condition, e.g.. mRNA encoding stromelysin, and in particular, those mRNA targets disclosed in the accompanying tables, which include both hammerhead and hairpin target sites.
• the site is flanked by regions to which appropriate substrate binding arms can be synthesized and an appropriate hammerhead or hairpin motif can be added to provide enzymatic activity, by methods described herein and known in the art.
• arms I and III are modified to be specific substrate-binding arms, and arm II remains essentially as shown.
• Ribozymes that cleave stromelysin mRNAs represent a novel therapeutic approach to arthritic disorders like osteoarthritis.
• the invention features use of ribozymes to treat osteoarthritis, e.g.. by inhibiting the synthesis of prostromelysin molecule in synovial cells or by the inhibition of matrix metalloproteinases.
• ribozymes are able to inhibit the secretion of stromelysin and that the catalytic activity of the ribozymes is required for their inhibitory effect.
• Those of ordinary skill in the art will find that it is clear from the examples described that other ribozymes that cleave stromelysin encoding mRNAs may be readily designed and are within the invention.
• the invention features a mammalian cell which includes an enzymatic RNA molecule as described above.
• the mammalian cell is a human cell; and the invention features an expression vector which includes nucleic acid encoding an enzymatic RNA molecule described above, located in the vector, e.g.. in a manner which allows expression of that enzymatic RNA molecule within a mammalian cell; or a method for treatment of a disease or condition by administering to a patient an enzymatic RNA molecule as described above.
• the invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the mRNA causative of an arthritic condition.
• Such enzymatic RNA molecules can be delivered exogenously or endogenously to infected cells.
• the small size (less than 40 nucleotides, preferably between 32 and 36 nucleotides in length) of the molecule allows the cost of treatment to be reduced.
• the enzymatic RNA molecules of this invention can be used to treat arthritic or prearthritic conditions. Such treatment can also be extended to other related genes in nonhuman primates. Affected animals can be treated at the time of arthritic risk detection, or in a prophylactic manner. This timing of treatment will reduce the chance of further arthritic damage.
• the invention features novel nucleic acid-based techniques [e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups (Cook et al., U.S. Patent 5,359,051 )] and methods for their use to induce graft tolerance, to treat autoimmune diseases such as lupus, rheumatoid arthritis, multiple sclerosis and to treatment of allergies.
• enzymatic nucleic acid molecules e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups (Cook et al., U.S. Patent 5,359,051 )
• the invention features use of one or more of the nucleic acid-based techniques to induce graft tolerance by inhibiting the synthesis of B7-1 , B7-2, B7-3 and CD40 proteins.
• inhibitor is meant that the activity of B7-1 , B7-2, B7-3 and/or CD40 or level of mRNAs encoded by B7-1 , B7-2, B7-3 and or CD40 is reduced below that observed in the absence of the nucleic acid.
• inhibition with ribozymes preferably is below that level observed in the presence of an enzymatically inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
• RNA to B7-1 , B7-2, B7-3 and/or CD40 is meant to include those naturally occurring RNA molecules associated with graft rejection in various animals, including human, mice, rats, rabbits, primates and pigs.
• antisense nucleic acid is meant a non-enzymatic nucleic acid molecule that binds to another RNA (target RNA) by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261 , 1004).
• target RNA RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261 , 1004).
• 2-5A antisense chimera 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. NatI. Acad. Sci. USA 90, 1300).
• triplex DNA an oligonucleotide that can bind to a double- stranded DNA in a sequence-specific manner to form a triple-strand helix. Triple-helix formation has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. NatI. Acad. Sci. USA 89, 504).
• RNA RNA
• Ribozymes that cleave the specified sites in B7-1 , B7-2, B7-3 and/or
• CD40 mRNAs represent a novel therapeutic approach to induce graft tolerance and treat autoimmune diseases, allergies and other inflammatory conditions. Applicant indicates that ribozymes are able to inhibit the activity of B7-1 , B7-2, B7-3 and/or CD40 and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave these sites in B7-1 , B7-2, B7-3 and/or CD40 mRNAs may be readily designed and are within the invention.
• 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 B7-1 , B7-2, B7-3 and/or CD40 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.
• the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.
• 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 levels of B7-1 , B7-2, B7-3 and/or CD40 activity in a cell or tissue.
• diseases or conditions that are related to the levels of B7-1 , B7-2, B7-3 and/or CD40 activity in a cell or tissue.
• related is meant that the inhibition of B7-1 , B7-2, B7-3 and/or CD40 mRNAs and thus reduction in the level respective 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.
• the ribozymes have binding arms which are complementary to the sequences in Tables Bll, BIV, BVI, BVIII, BX, BXII, BXIV, BXV, BXVI, BXVII, BXVIII and BXIX.
• ribozymes examples include ribozymes, ribozymes, and ribozymes.
• ribozymes consist essentially of sequences defined in these Tables.
• ribozymes that cleave target molecules and inhibit B7-1 , B7-2, B7-3 and/or CD40 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.
• the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells.
• viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary.
• 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.
• Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art.
• Stem II can be > 2 base-pairs long.
• Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art
• Figure 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987. Nature. 327, 596-600) into a substrate and enzyme portion
• Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature , 334, 585- 591 ) into two portions
• Figure 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 > 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.
• 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 > 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.
• " - " refers to a chemical bond.
• Figure 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art.
• Figure 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.
• Figure 6 is a schematic representation of an RNaseH accessibility assay. Specifically, the left side of Figure 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 Figure 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.
• Figure 7 shows in vitro cleavage of stromelysin mRNA by HH ribozymes.
• Figure 8 shows inhibition of stromelysin expression by 21 HH ribozyme in HS-27 fibroblast cell line.
• Figure 9 shows inhibition of stromelysin expression by 463HH ribozyme in HS-27 fibroblast cell line.
• Figure 10 shows inhibition of stromelysin expression by 1049HH ribozyme in HS-27 fibroblast cell line.
• Figure 11 shows inhibition of stromelysin expression by 1366HH ribozyme in HS-27 fibroblast cell line.
• Figure 12 shows inhibition of stromelysin expression by 141 OHH ribozyme in HS-27 fibroblast cell line.
• Figure 13 shows inhibition of stromelysin expression by 1489HH ribozyme in HS-27 fibroblast cell line.
• Figure 14 shows 1049HH ribozyme-mediated reduction in the level of stromelysin mRNA in rabbit knee.
• Figure 15 shows 1049HH ribozyme-mediated reduction in the level of stromelysin mRNA in rabbit knee.
• Figure 16 shows 1049HH ribozyme-mediated reduction in the level of stromelysin mRNA in rabbit knee.
• Figure 17 shows the effect of phosphorothioate substitutions on the catalytic activity of 1049 2'-C-allyl HH ribozyme.
• ribozyme contains phosphorothioate substitutions.
• Figure 18 is a diagrammatic representation of chemically modified ribozymes targeted against stromelysin RNA.
• Figures 22a-d are diagrammatic representations of non-limiting examples of base modifications for adenine, guanine, cytosine and uracil, respectively.
• Figure 23 is a diagrammatic representation of a position numbered hammerhead ribozyme (according to Hertel et al., Nucleic Acids Res. 1992, 20:3252) showing specific substitutions in the catalytic core and substrate binding arms. Compounds 4, 9, 13, 17, 22 and 23 are described in Fig. 24.
• Figure 24 is a diagrammatic representation of various nucleotides that can be substituted in the catalytic core of a hammerhead ribozyme.
• Figure 25 is a diagrammatic representation of the synthesis of a ribothymidine phosphoramidite.
• Figure 26 is a diagrammatic representation of the synthesis of a 5-methylcytidine phosphoramidite.
• Figure 27 is a diagrammatic representation of the synthesis of 5-bromouridine phosphoramidite.
• Figure 28 is a diagrammatic representation of the synthesis of
• Figure 29 is a diagrammatic representation of the synthesis of 2,6-diaminopurine phosphoramidite.
• Figure 30 is a diagrammatic representation of the synthesis of a 6-methyluridine phosphoramidite.
• Figure 31 is a representation of a hammerhead ribozyme targeted to site A (HH-A). Site of 6-methyl U substitution is indicated.
• Figure 32 shows RNA cleavage reaction catalyzed by HH-A ribozyme containing 6-methyl U-substitution (6-methyl-U4).
• U4 represents a HH-A ribozyme containing no 6-methyl-U substitution.
• Figure 33 is a representation of a hammerhead ribozyme targeted to site B (HH-B). Sites of 6-methyl U substitution are indicated.
• Figure 34 shows RNA cleavage reaction catalyzed by HH-B ribozyme containing 6-methyl U-substitutions at U4 and U7 positions (6-methyl-U4).
• U4 represents a HH-B ribozyme containing no 6-methyl-U substitution.
• Figure 35 is a representation of a hammerhead ribozyme targeted to site C (HH-C). Sites of 6-methyl U substitution are indicated.
• Figure 36 shows RNA cleavage reaction catalyzed by HH-C ribozyme containing 6-methyl U-substitutions at U4 and U7 positions (6-methyl-U4).
• U4 represents a HH-C ribozyme containing no 6-methyl-U substitution.
• Figure 37 shows 6-methyl-U-substituted HH-A ribozyme-mediated inhibition of rat smooth muscle cell proliferation.
• Figure 38 shows 6-methyl-U-substituted HH-C ribozyme-mediated inhibition of stromelysin protein production in human synovial fibroblast cells.
• Figure 39 is a diagrammatic representation of the synthesis of pyridin-2- one nucleoside and pyridin-4-one nucleoside phosphoramidite.
• Figure 40 is a diagrammatic representation of the synthesis of 2-O-f-
• Figure 41 is a diagrammatic representation of the synthesis of pseudouridine, 2,4,6-trimethoxy benzene nucleoside and 3-methyluridine phosphoramidite.
• Figure 42 is a diagrammatic representation of the synthesis of dihydrouridine phosphoramidite.
• Figure 43 A) is diagrammatic representation of a hammerhead ribozyme targeted to site B.
• B) shows RNA cleavage reaction catalyzed by hammerhead ribozyme with modified base substitutions at either position 4 or position 7.
• Figure 44 shows further kinetic characterization of RNA cleavage reactions catalyzed by HH-B ribozyme (A); HH-B with pyridin-4-one substitution at position 7 (B); and HH-B with phenyl substitution at position 7 (C).
• Figure 45 is a diagrammatic representation of the synthesis of 2-O-f- Butyldimethylsilyl-5-0-Dimethoxytrityl-3-0-(2-Cyanoethyl- ⁇ /, ⁇ /- diisopropylphosphoramidite)-1-Deoxy-1-Naphthyl- ⁇ -D-Ribofuranose.
• Figure 46 is a diagrammatic representation of the synthesis of Synthesis of 2-0- -Butyldimethylsilyl-5-0-Dimethoxytrityl-3-0-(2-Cyanoethyl- ⁇ /, V- diisopropylphosphoramidite)-1-Deoxy-1 -(p-Aminophenyl)- ⁇ -D-Ribofuranose.
• Figure 47 is a diagrammatic representation of a position numbered hammerhead ribozyme (according to Hertel et al. Nucleic Acids Res. 1992, 20, 3252) showing specific substitutions.
• Figure 48 shows the structures of various 2'-alkyl modified nucleotides which exemplify those of this invention.
• R groups are alkyl groups, Z is a protecting group.
• Figure 49 is a diagrammatic representation of the synthesis of 2'-C-allyl uridine and cytidine.
• Figure 50 is a diagrammatic representation of the synthesis of 2'-C- methylene and 2'-C-difluoromethylene uridine.
• Figure 51 is a diagrammatic representation of the synthesis of 2'-C- methylene and 2'-C-difluoromethylene cytidine.
• Figure 52 is a diagrammatic representation of the synthesis of 2'-C- methylene and 2'-C-difluoromethylene adenosine.
• Figure 53 is a diagrammatic representation of the synthesis of 2'-C- carboxymethylidine uridine, 2'-C-methoxycarboxymethylidine uridine and derivatized amidites thereof.
• X is CH3 or alkyl as discussed above, or another substituent.
• Figure 54 is a diagrammatic representation of the synthesis of 2'-C-allyl uridine and cytidine phosphoramidites.
• Figure 55 is a diagrammatic representation of the synthesis of 2'-0- alkylthioalkyl nucleosides or non-nucleosides and their phosphoramidites.
• R is an alkyl as defined above.
• B is any naturally occuring or modified base bearing any N-protecting group suitable for standard oligonucleotide synthesis (Usman et al., supra; Scaringe et al., supra), and/or H (non- nucleotide), as described by the publications discussed above.
• CE is cyanoethyl
• DMT is a standard blocking group. Other abbreviations are standard in the art.
• Figure 56 is a diagrammatic representation of a hammerhead ribozyme, targeted to site B (HH-B), containing 2'-0-methylthiomethyl substitutions.
• Figure 57 shows RNA cleavage activity catalyzed by 2'-0- methylthiomethyl substituted ribozymes.
• a plot of percent cleaved as a function of time is shown. The reactions were carried out at 37°C in the presence of 40 nM ribozyme, 1 nM substrate and 10 mM MgCl2-
• Control HH- B ribozyme contained the following modifications; 29 positions were modified with 2'-0-methyl, U4 and U7 positions were modified with 2'-amino groups, 5 positions contained 2'-OH groups. These modifications of the control ribozyme have previously been shown not to significantly effect the activity of the ribozyme (Usman et al., 1994 Nucleic Acids Symposium Series 31 , 163).
• Figure 58 is a diagrammatic representation of the synthesis of an abasic deoxyribose or ribose non-nucleotide mimetic phosphoramidite.
• Figure 59 is a diagrammatic representation of a hammerhead ribozyme targeted to site B (HH-B). Arrow indicates the cleavage site.
• Figure 60 is a diagrammatic representation of HH-B ribozyme containing abasic substitutions (HH-Ba) at various positions. Ribozymes were synthesized as described in the application. "X" shows the positions of abasic substitutions. The abasic substitutions were either made individually or in certain combinations.
• Figure 61 shows the in vitro RNA cleavage activity of HH-B and HH-Ba ribozymes. All RNA, refers to HHA ribozyme containing no abasic substitution.
• U4 Abasic refers to HH-Ba ribozyme with a single abasic (ribose) substitution at position 4.
• U7 Abasic refers to HH-Ba ribozyme with a single abasic (ribose) substitution at position 7.
• Figure 62 shows in vitro RNA cleavage activity of HH-B and HH-Ba ribozymes.
• Abasic Stem II Loop refers to HH-Ba ribozyme with four abasic (ribose) substitutions within the loop in stem II.
• Figure 63 shows in vitro RNA cleavage activity of HH-B and HH-Ba ribozymes.
• 3'-lnverted Deoxyribose refers to HH-Ba ribozyme with an inverted deoxyribose (abasic) substitution at its 3' termini.
• Figure 64 is a diagrammatic representation of a hammerhead ribozyme targeted to site A (HH-A).
• Target A is involved in the proliferation of mammalian smooth muscle cells. Arrow indicates the site of cleavage.
• Inactive version of HH-A contains 2 base-substitutions (G5U and A15.1 U) that renders the ribozyme catalytically inactive.
• Figure 65 is a diagrammatic representation of HH-A ribozyme with abasic substitution (HH-Aa) at position 4.
• X shows the position of abasic substitution.
• Figure 66 shows ribozyme-mediated inhibition of rat aortic smooth muscle cell (RASMC) proliferation.
• RASMC rat aortic smooth muscle cell
• Figure 67 is a schematic representation of a two pot deprotection protocol with ethylamine (EA).
• Figure 68 shows a strategy used in synthesizing a hammerhead ribozyme from two halves.
• X and Y represent reactive moieties that can undergo a chemical reaction to form a covalent bond (represented by the solid curved line).
• Figure 69 shows various non-limiting examples of reactive moieties that can be placed in the nascent loop region to form a covalent bond to provide a full-length ribozyme.
• CH2 can be any linking chain as described above including groups such as methylenes, ether, ethylene glycol, thioethers, double bonds, aromatic groups and others; each n independently is an integer from 0 to 10 inclusive and may be the same or different; each R independently is a proton or an alkyl, alkenyl and other functional groups or conjugates such as peptides, steroids, hoemones, lipids, nucleic acid sequences and others that provides nuclease resistance, improved cell association, improved cellular uptake or interacellular localization.
• Figure 70 shows non-limiting examples of covalent bonds that can be formed to provide the full length ribozyme.
• the morpholino group arises from reductive reaction of a dialdehyde, which arises from oxidative cleavage of a ribose at the 3'-end of one half ribozyme with an amine at the 5'-end of the half ribozyme.
• the amide bond is produced when an acid at the 3'-end of one half ribozyme is coupled to an amine at the 5'-end of the other half ribozyme.
• Figure 71 shows non-limiting examples of three ribozymes that were synthesized from coupling reactions of two halves. All three were targeted to the site A of c-myb RNA (HH-A).
• HH-A1 was formed from the reaction of two thiols to provide the disulfide linked ribozyme.
• HH-A2 and HH-A3 were formed using the morpholino reaction.
• HH-A2 contains a five atom spacer linking the terminal amine to the 5'-end of the half ribozyme.
• HH-A3 contains a six carbon spacer linking the terminal amine to the 5'-end of the half ribozyme.
• Figure 72 shows comparative cleavage activity of half ribozymes, containing five and six base pair stem II regions, that are not covalently linked vs a full length ribozyme. Assays were carried out under ribozyme excess conditions.
• Figure 73 shows comparative cleavage activity of half ribozymes, containing seven and eight base pair stem II regions, that are not covalently linked vs a full length ribozyme. Assays were carried out under ribozyme excess conditions.
• Figure 74 shows comparative cleavage assay of HH-A1 , HH-A2 and HH- A3 (see Figure 72) formed from crosslinking reactions vs a full length ribozyme control. Assays were carried out under ribozyme excess conditions.
• Figure 75 Schematic representation of RNA polymerse III promoter structure. Arrow indicates the transcription start site and the direction of coding region.
• A, B and C refer to consensus A, B and C box promoter sequences.
• I refers to intermediate cis-acting promoter sequence.
• PSE refers to proximal sequence element.
• DSE refers to distal sequence element.
• ATF refers to activating transcription factor binding element.
• ? refers to cis- acting sequence element that has not been fully characterized.
• EBER Epstein-Barr-virus-encoded-RNA. TATA is a box well known in the art.
• Figure 76 is a general formula for pol III RNA of this invention.
• Figure 77 is a diagrammatic representation of a U6-S35 Chimera. The S35 motif and the site of insertion of a desired RNA are indicated. This chimeric RNA transcript is under the control of a U6 small nuclear RNA (snRNA) promoter.
• snRNA small nuclear RNA
• Figure 78 is a diagrammatic representation of a U6-S35-ribozyme chimera.
• the chimera contains a hammerhead ribozyme targeted to site I (HHI).
• Figure 79 is a diagrammatic representation of a U6-S35-ribozyme chimera.
• the chimera contains a hammerhead ribozyme targeted to site II (HHII).
• Figure 80 shows RNA cleavage reaction catalyzed by a synthetic hammerhead ribozyme (HHI) and by an in vitro transcript of U6-S35-HHI hammerhead ribozyme.
• HHI synthetic hammerhead ribozyme
• Figure 81 shows stability of U6-S35-HHII RNA transcript in 293 mammalian cells as measured by actinomycin D assay.
• Figure 82 is a diagrammatic representation of an adenovirus VA1 RNA. Various domains within the RNA secondary structure are indicated.
• Figure 83 A shows a secondary structure model of a VA1-S35 chimeric RNA containing the promoter elements A and B box. The site of insertion of a desired RNA and the S35 motif are indicated. The transcription unit also contains a stable stem (S35-like motif) in the central domain of the VA1 RNA which positions the desired RNA away from the main transcript as an independent domain.
• 83B shows a VA1 -chimera which consists of the terminal 75 nt of a VA1 RNA followed by the HHI ribozyme.
• Figure 84 shows a comparison of stability of VA1 -chimeric RNA vs VA1- S35-chimeric RNA as measured by actinomycin D assay.
• VA1 -chimera consists of terminal 75 nt of VA1 RNA followed by HHI ribozyme.
• VA1-S35- chimera structure and sequence is shown in Figure 83.
• Ribozymes in one aspect of this invention block to some extent stromelysin expression and can be used to treat disease or diagnose such disease. Ribozymes are delivered to cells in culture and to cells or tissues in animal models of osteoarthritis (Hembry et al., 1993 Am. J. Pathol. 143, 628). Ribozyme cleavage of stromelysin encoding mRNAs in these systems may prevent inflammatory cell function and alleviate disease symptoms.
• ribozymes of this invention block to some extent B7-1 , B7-2, B7-3 and/or CD40 production and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture, to cells or tissues in animal models of transplantation, autoimmune diseases and/or allergies and to human cells or tissues ex vivo or in vivo. Ribozyme cleavage of B7-1 , B7-2 and/or CD40 encoded mRNAs in these systems may alleviate disease symptoms.
• 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., WO 95/13380 and Stinchcomb et al WO 95/23225. 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, rabbit and other animal RNA are provided, those in the art will recognize that the equivalent human RNA targets described can be used as described below.
• the same target may be used, but binding arms suitable for targeting human RNA sequences are present in the ribozyme.
• Such targets may also be selected as described below.
• the sequence of human and rabbit stromelysin mRNA were screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables All, AMI, AIV, AVI, AVIII and AIX (All sequences are 5' to 3' in the tables.). While rabbit and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, rabbit targeted ribozymes are 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.
• CD40 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 Bll, BIV, BVI, BVIII, BX, BXII, BXIV, BXV, BXVI, BXVII, BXVIII and BXIX (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 mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility.
• mouse 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 are designed that could bind and are individually analyzed by computer folding (Jaeger et al., 1989 Proc. NatI. 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 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.
• RNA is screened for accessible cleavage sites by the method described generally in Draper WO 93/23569. Briefly, DNA oligonucleotides representing potential hammerhead or hairpin ribozyme cleavage sites are synthesized. A polymerase chain reaction is used to generate a substrate for T7 RNA polymerase transcription from human or rabbit stromelysin cDNA clones. Labeled RNA transcripts are synthesized in vitro from the two templates. The oligonucleotides and the labeled transcripts are annealed, RNaseH is added and the mixtures are incubated for the designated times at 37°C. Reactions are stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved is determined by autoradiographic quantitation using a Phosphorlmaging system. From these data, hammerhead ribozyme sites are chosen as the most accessible.
• Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message.
• the binding arms are complementary to the target site sequences described above.
• the ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc. 109, 7845-7854 and in Scaringe et al., 1990 Nucleic Acids Res.. 18, 5433-5441 ; Wincott et al., 1995 Nucleic Acids Res.
• Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res.. 20, 3252) . Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res.. 20, 2835-2840).
• ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702).
• Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Stinchcomb et al, supra) and are resuspended in water.
• stem loop II sequence of hammerhead ribozymes listed in Tables AV and AVII can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form.
• stem-loop AIV sequence of hairpin ribozymes listed in Tables AVI and AVII (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.
• sequences listed in Tables AV, AVII, AVIII and AIX may be formed of ribonucleotides or other nucleotides or non-nucleotides.
• Such ribozymes are equivalent to the ribozymes described specifically in the Tables.
• Ribozyme activity can be optimized as described by Stinchcomb et al., supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms (stems I and III, see Figure 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.
• 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.
• ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
• the RNA/vehicle combination is locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent.
• 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.
• the ribozyme is administered to the site of B7-1 , B7-2, B7-3 and/or CD40 expression (APC) in an appropriate liposomal vesicle.
• APCs isolated from donor are treated with the ribozyme preparation (or other nucleic acid therapeutics) ex vivo and the treated cells are infused into recipient.
• cells, tissues or organs are directly treated with nucleic acids of the present invention prior to transplantation into a recipient.
• RNA polymerase I RNA polymerase I
• RNA polymerase II RNA polymerase II
• RNA polymerase III RNA polymerase 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. NatI. Acad. Sci. U S A. 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).
• ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev..
• 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 vectors), or viral RNA vectors (such as retroviral or alphavirus vectors).
• plasmid DNA vectors such as adenovirus or adeno- associated vectors
• viral RNA vectors such as retroviral or alphavirus vectors
• a transcription unit expressing a ribozyme that cleaves stromelysin RNA is inserted into a plasmid DNA vector or an adenovirus DNA virus or adeno-associated virus (AAV) vector.
• AAV adeno-associated virus
• Both viral vectors have been used to transfer genes to the lung and both vectors lead to transient 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.
• nucleotides comprise a base, sugar and a monophosphate group. Accordingly, nucleotide derivatives or modifications may be made at the level of the base, sugar or monophosphate groupings Bases may be substituted with various groups, such as halogen, hydroxy, amine, alkyl, azido, nitro, phenyl and the like.”
• W093/23569, WO95/06731 , WO95/04818, and W095/133178 describe various modifications that can be introduced into ribozyme structures.
• This invention relates to production of enzymatic RNA molecules or ribozymes having enhanced or reduced binding affinity and enhanced enzymatic activity for their target nucleic acid substrate by inclusion of one or more modified nucleotides in the substrate binding portion of a ribozyme such as a hammerhead, hairpin, VS ribozyme or hepatitis delta virus derived ribozyme.
• a ribozyme such as a hammerhead, hairpin, VS ribozyme or hepatitis delta virus derived ribozyme.
• Applicant has recognized that only small changes in the extent of base-pairing or hydrogen bonding between the ribozyme and substrate can have significant effect on the enzymatic activity of the ribozyme on that substrate.
• a subtle alteration in the extent of hydrogen bonding along a substrate binding arm of a ribozyme can be used to improve the ribozyme activity compared to an unaltered ribozyme containing no such altered nucleotide.
• a guanosine base may be replaced with an inosine to produce a weaker interaction between a ribozyme and its substrate, or a uracil may be replaced with a bromouracil (BrU) to increase the hydrogen bonding interaction with an adenosine.
• BrU bromouracil
• Other examples of alterations of the four standard ribonucleotide bases are shown in Figures 22a-d with weaker or stronger hydrogen bonding abilities shown in each figure.
• nucleotides maintains or enhances enzymatic activity compared to an unmodified molecule.
• nucleotides are noted in Figure 23.
• the preferred sequence of a hammerhead ribozyme in a 5' to 3' direction of the catalytic core is CUG ANG A G » C GAA A, wherein N can be any base or may lack a base (abasic); G » C is a base-pair.
• the nature of the base-paired stem II ( Figures 1 , 2 and 23) and the recognition arms of stems I and III are variable.
• base-modified nucleotides in those regions that maintain or enhance the catalytic activity and/or the nuclease resistance of the hammerhead ribozyme are described. (Bases which can be modified include those shown in capital letters).
• bases where none of the functional groups are important in the complexing of magnesium or other functions of a ribozyme they are optionally replaced with a purine ribonucleoside ( Figure 24, 23), which significantly reduces the complexity of chemical synthesis of ribozymes, as no base-protecting group is required during chemical incorporation of the purine nucleus.
• base-modified nucleotides may be used to enhance the specificity or strength of binding of the recognition arms with similar modifications.
• Base-modified nucleotides in general, may also be used to enhance the nuclease resistance of the catalytic nucleic acids in which they are incorporated.
• the invention provides ribozymes having increased enzymatic activity in vitro and in vivo as can be measured by standard kinetic assays.
• the kinetic features of the ribozyme are enhanced by selection of appropriate modified bases in the substrate binding arms.
• Applicant recognizes that while strong binding to a substrate by a ribozyme enhances specificity, it may also prevent separation of the ribozyme from the cleaved substrate.
• applicant provides means by which optimization of the base pairing can be achieved.
• the invention features ribozymes with modified bases with enzymatic activity at least 1.5 fold (preferably 2 or 3 fold) or greater than the unmodified corresponding ribozyme.
• the invention also features a method for optimizing the kinetic activity of a ribozyme by introduction of modified bases into a ribozyme and screening for those with higher enzymatic activity. Such selection may be in vitro or in vivo. By enhanced activity is meant to include activity measured in vivo where the activity is a reflection of both catalytic activity and ribozyme stability. In this invention, the product of these properties in increased or not significantly (less that 10 fold) decreased in vivo compared to an all RNA ribozyme.
• zymatic portion is meant that part of the ribozyme essential for cleavage of an RNA substrate.
• substrate binding arm is meant that portion of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired.
• Such arms are shown generally in Figures 1-3 as discussed below. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions; e.g., ribozyme sequences within stems I and III of a standard hammerhead ribozyme make up the substrate-binding domain (see Figure 1 ).
• unmodified nucleotide base is meant one of the bases adenine, cytosine, guanosine, uracil joined to the 1 ' carbon of ⁇ -D-ribo-furanose.
• the sugar also has a phosphate bound to the 5' carbon.
• These nucleotides are bound by a phosphodiester between the 3' carbon of one nucleotide and the 5' carbon of the next nucleotide to form RNA.
• modified nucleotide base any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base which has an effect on the ability of that base to hydrogen bond with its normal complementary base, either by increasing the strength of the hydrogen bonding or by decreasing it (e.g., as exemplified above for inosine and bromouracil).
• modified bases include those shown in Figures 22a-d and other modifications well known in the art, including heterocyclic derivatives and the like.
• the modified ribozyme is a hammerhead, hairpin VS ribozyme or hepatitis delta virus derived ribozyme, and the hammerhead ribozyme includes between 32 and 40 nucleotide bases.
• the selection of modified bases is most preferably chosen to enhance the enzymatic activity (as observed in standard kinetic assays designed to measure the kinetics of cleavage) of the selected ribozyme, i.e., to enhance the rate or extent of cleavage of a substrate by the ribozyme, compared to a ribozyme having an identical nucleotide base sequence without any modified base.
• kinetic assays or “kinetics of cleavage” is meant an experiment in which the rate of cleavage of target RNA is determined. Often a series of assays are performed in which the concentrations of either ribozyme or substrate are varied from one assay to the next in order to determine the influence of that parameter on the rate of cleavage.
• rate of cleavage is meant a measure of the amount of target RNA cleaved as a function of time.
• Enzymatic nucleic acid having a hammerhead configuration and modified bases which maintain or enhance enzymatic activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid.
• modified bases in this aspect is meant those shown in Figure 22 A-D, and 24, 30, and 42B or their equivalents; such bases may be used within the catalytic core of the enzyme as well as in the substrate-binding regions.
• the invention features modified ribozymes having a base substitution selected from pyridin-4-one, pyridin-2- one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and aminophenyl.
• substitution in the core may decrease in vitro activity but enhances stability. Thus, in vivo the activity may not be significantly lowered.
• such ribozymes are useful in vivo even if active over all is reduced 10 fold.
• Such ribozymes herein are said to "maintain" the enzymatic activity on all RNA ribozyme.
• RNA deprotection of the RNA was performed as follows.
• the base-deprotected oiigoribonucleotide was resuspended in anhydrous TEA»HF/NMP solution (250 ⁇ L of a solution of 1.5mL ⁇ /-methylpyrrolidinone, 750 ⁇ L TEA and 1.0 mL TEA « 3HF to provide a 1.4M HF concentration) and heated to
• the TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB (10 mL), the RNA was eluted with 2 M TEAB (10 mL) and dried down to a white powder.
• Qiagen 500® anion exchange cartridge Qiagen Inc.
• Inactive hammerhead ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from (Hertel, K. J., et al., 1992, Nucleic Acids Res.. 20, 3252)).
• Hairpin ribozymes are synthesized either as one part or in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840).
• Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Stinchcomb et al., International PCT Publication No. WO 95/23225, and are resuspended in water.
• HPLC high pressure liquid chromatography
• ribozyme structure can be made to enhance the utility of ribozymes. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such ribozymes to the target site, e.g.. to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells. Examples of such ribozymes are provided in Usman et al., WO 95/13378 and below.
• This invention relates to the use of 2'-deoxy-2'-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides.
• 2'-deoxy-2'-alkylnucleotide-containing enzymatic nucleic acids are catalytic nucleic acid molecules that contain 2'-deoxy-2'- alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences.
• RNA or DNA molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner.
• Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired.
• Such enzymatic molecules can be targeted to virtually any RNA transcript.
• 2'-deoxy-2'-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides. Contrary to the findings of De Mesmaeker et al. applicant has found that such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of the 2'-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an essential base pair forming region then the enhanced stability that it provides to the molecule is advantageous.
• the enhanced stability may make the loss of activity of less consequence.
• a 2'-deoxy-2'-alkyl-containing molecule has 10% the activity of the unmodified molecule, but has 10-fold higher stability in vivo then it has utility in the present invention.
• antisense oligonucleotides containing such modifications The same analysis is true for antisense oligonucleotides containing such modifications.
• the invention also relates to novel intermediates useful in the synthesis of such nucleotides and oligonucleotides (examples of which are shown in the Figures 48-54), and to methods for their synthesis.
• the invention features 2'-deoxy-2'-alkylnucleotides, that is a nucleotide base having at the 2'-position on the sugar molecule an alkyl moiety and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the invention preferably includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above.
• each R group is any alkyl.
• an "alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups.
• the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
• alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.
• the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
• alkyl also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.
• the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
• alkyl does not include alkoxy groups which have an "-0-alkyl” group, where "alkyl” is defined as described above, where the O is adjacent the 2'-position of the sugar molecule.
• alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.
• An "aryl” group refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
• the preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
• alkylaryl refers to an alkyl group (as described above) covalently joined to an aryl group (as described above.
• Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
• Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.
• Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
• An "amide” refers to an -C(0)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
• An “ester” refers to an -C(0)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
• the invention features oligonucleotides having one or more 2'-deoxy-2'-alkylnucleotides (preferably not a 2'-alkyl- uridine or thymidine); e.g. enzymatic nucleic acids having a 2'-deoxy-2'-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 2'-position an alkyl group.
• the invention features 2'-deoxy-2'-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.
• the 2'-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall activity in vivo. Below are provided assays to determine which such molecules are useful.
• the invention features hammerhead motifs having enzymatic activity having ribonucleotides at locations shown in Figure 47 at 5, 6, 8, 12, and 15.1 , and having substituted ribonucleotides at other positions in the core and in the substrate binding arms if desired.
• core refers to positions between bases 3 and 14 in Figure 47, and the binding arms correspond to the bases from the 3'-end to base 15.1 , and from the 5'-end to base 2).
• Applicant has found that use of ribonucleotides at these five locations in the core provide a molecule having sufficient enzymatic activity even when modified nucleotides are present at other sites in the motif.
• Other such combinations of useful ribonucleotides can be determined as described by Usman et al. supra.
• This invention relates to the incorporation of 2'-0-alkyllthioalkyl and/or 2'- C-alkylthioalkyl nucleotides or non-nucleotides into nucleic acids, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides.
• 2'-0-alkylthioalkyl and/or 2'-C- alkylthioalkyl nucleotide or non-nucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 2'-0-alkylthioalkyl and/or 2'-C- alkylthioalkyl nucleotide or non-nucleotides components replacing one or more bases or regions including, but not limited to, those bases in double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences.
• RNA or DNA molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner.
• Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired.
• Such enzymatic molecules can be targeted to virtually any RNA transcript.
• 2'-0-alkylthioalkyl and/or 2'-C-alkylthioalkyl nucleotides or non-nucleotides which may be present in enzymatic nucleic acid or in antisense oligonucleotides or 2-5A antisense chimera.
• Such nucleotides or non-nucleotides are useful since they enhance the activity of the antisense or enzymatic molecule.
• the invention also relates to novel intermediates useful in the synthesis of such nucleotides or non-nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis.
• the invention features 2'-0-alkylthioalkyl nucleosides or non- nucleosides, that is a nucleoside or non-nucleosides having at the 2'-position on the sugar molecule a 2'-0-alkylthioalkyl moiety.
• the invention also features 2'-0-alkylthioalkyl nucleotides or non-nucleotides. That is, the invention preferably includes those nucleotides or non-nucleotides having 2' substitutions as noted above useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above.
• non-nucleotide refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
• the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenine, guanine, cytosine, uracil or thymine. It may have substitutions for a 2' or 3' H or OH as described in the art. See Eckstein et al. and Usman et al., supra.
• nucleotide refers to the regular nucleotides (A, U, G, T and C) and modified nucleotides such as 6-methyl U, inosine, 5-methyl C and others.
• nucleotide is used as recognized in the art to include natural bases, and modified bases well known in the art. Such bases are generally located at the 1 1 position of a sugar moiety.
• non- nucleotide as used herein to encompass sugar moieties lacking a base or having other chemical groups in place of a base at the V position.
• Such molecules generally include those having the general formula:
• R1 represents 2'-0-alkylthioalkyl or 2'-C-alkylthioalkyl
• X represents a base or H
• Y represents a phosphorus-containing group
• R2 represents H, DMT or a phosphorus-containing group ( Figure 55).
• Phosphorus-containing group is generally a phosphate, thiophosphate, H-phosphonate, methylphosphonate, phosphoramidite or other modified group known in the art.
• the invention features 2'-C-alkylthioalkyl nucleosides or non-nucleosides, that is a nucleotide or a non-nucleotide residue having at the 2'-position on the sugar molecule a 2'-C-alkylthioalkyl moiety.
• the invention also features 2'-C-alkylthioalkyl nucleotides or non- nucleotides. That is, the invention preferably includes all those 2' modified nucleotides or non-nucleotides useful for making enzymatic nucleic acids or antisense molecules as described above that are not described by the art discussed above.
• an "alkyl” group is as defined above, except that the term includes 2'-0-alkyl moeities.
• the invention features oligonucleotides having one or more 2'-0-alkylthioalkyl and/or 2'-C- alkylthioalkyl nucleotides or non-nucleotides; e.g.
• enzymatic nucleic acids having a 2'-0-methylthiomethyl and/or 2'-C-alkylthioalkyl nucleotides or non- nucleotides ; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide or a non- nucleotide moiety having at its 2'-position an 2'-0-alkylthioalkyl and/or 2'-C- alkylthioalkyl group.
• the invention features 2'-0-alkylthioalkyl and/or 2'-C-alkylthioalkyl nucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.
• the 2'-0-alkylthioalkyl and/or 2'-C-alkylthioalkyl derivatives of this invention provide enhanced activity and stability to the oligonulceotides containing them.
• the invention features oligonucleotides having one or more 2'-0-alkylthioalkyl and/or 2'-C- alkylthioalkyl abasic (non-nucleotide) moeities.
• enzymatic nucleic acids having a 2'-0-alkylthioalkyl and/or 2'-C-alkylthioalkyl abasic moeity; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one position having at its 2'- position an 2'-0-alkylthioalkyl or 2'-C-alkylthioalkyl group.
• the invention features enzymatic nucleic acids containing one or more 2'-0-alkylthioalkyl and/or 2'-C-alkylthioalkyl substitutions either in the enzymatic portion, substrate binding portion or both, as long as the catalytic activity of the ribozyme is not significantly decreased.
• the invention features the use of 2'-
• O-alkylthioalkyl moieties as protecting groups for 2'-hydroxyl positions of ribofuranose during nucleic acid synthesis.
• Figure 1 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided.
• the preferred sequence of a hammerhead ribozyme in a 5'- to 3'-direction of the catalytic core is CUGANGAG [base paired with] CGAAA.
• CUGANGAG base paired with
• the use of 2'-0-alkylthioalkyl and/or 2'-C- alkylthioalkyl substituted nucleotides or non-nucleotides that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Substitutions of any nucleotide with any of the modified nucleotides or non-nucleotides discussed above are possible.
• linkages between the building units of the polymeric chain may be linkages capable of bridging the units together for either in vitro or in vivo.
• the linkage may be a phosphorous containing linkage, e.g., phosphodiester or phosphothioate, or may be a nitrogen containing linkage, e.g., amide.
• the chimeric polymer may contain non-nucleotide spacer molecules along with its other nucleotide or analogue units. Examples of spacer molecules which may be used are described in Nielsen et al. Science. 254:1497-
• tem ll may be replaced, for example, with a linker selected from optionally substituted polyphosphodiester (such as poly(1-phospho-3- propanol)), optionally substituted alkyl, optionally substituted polyamide, optionally substituted glycol, and the like.
• optionally substituted polyphosphodiester such as poly(1-phospho-3- propanol)
• optionally substituted alkyl optionally substituted polyamide, optionally substituted glycol, and the like.
• Optional substituents are well known in the art, and include alkoxy (such as methoxy, ethoxy and propoxy), straight or branch chain lower alkyl such as Ci - C5 alkyl), amine, aminoalkyl (such as amino C1 - C5 alkyl), halogen (such as F,
• suitable linkers may comprise polycyclic molecules, such as those containing phenyl or cyclohexyl rings.
• the linker (L) may be a polyether such as polyphosphopropanediol, polyethyleneglycol, a bifunctional polycyclic molecule such as a bifunctional pentalene, indene, naphthalene, azulene, heptalene, biphenylene, asymindacene, sym-indacene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, fluoranthene, acephenathrylene, aceanthrylene, triphenylene, pyrene, chrysene, naphthacene, thianthrene, isobenzofuran, chromene, xanthene, phenoxathiin, indolizine, isoindoie, 3-H-indole, indole, 1-H-indazole, 4-H-quinolizine, isoquinoline, quinoline, phthala
• the polycyclic molecule may be substituted of polysubstituted with C -C5 alkyl, alkenyl, hydroxyalkyl, halogen of haloalkyl group or with O-
• the linker is t e t r a p h 0 s p h o p r 0 p a n e d i o I o r pentaphosphopropanediol.
• polycyclic molecules there will be preferably 18 or more atoms bridging the nucleic acids. More preferably their will be from 30 to 50 atoms bridging, see for Example 5.
• the linker is a bifunctional carbazole or bifunctional carbazole linked to one or more polyphosphoropropanediol.
• Such compounds may also comprise suitable functional groups to allow coupling through reactive groups on nucleotides.
• This invention concerns the use of non-nucleotide molecules as spacer elements at the base of double-stranded nucleic acid (e.g., RNA or DNA) stems (duplex stems) or more preferably, in the single-stranded regions, catalytic core, loops, or recognition arms of enzymatic nucleic acids.
• Duplex stems are ubiquitous structural elements in enzymatic RNA molecules.
• a base or base-pair mimetic may be used to reduce the nucleotide requirement in the synthesis of such molecules, and to confer nuclease resistance (since they are non-nucleic acid components).
• abasic nucleotides i.e., moieties lacking a nucleotide base, but having the sugar and phosphate portions
• abasic nucleotides can be used to provide stability within a core of a ribozyme, e.g.. at U4 or N7 of a hammerhead structure shown in Figure 1.
• the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.
• non-nucleotide mimetics examples include those described by
• the enzymatic nucleic acid includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the non-nucleotide moiety.
• the enzymatic nucleic acid includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the non-nucleotide moiety.
• RNA Ribonucleic acid
• the necessary ribonucleotide components are known in the art, see, e.g., Usman, supra and Usman et al., Nucl. Acid. Svmp. Genes 31 :163. 1994.
• non-nucleotide-containing enzymatic nucleic acid means a nucleic acid molecule that contains at least one non-nucleotide component which replaces a portion of a ribozyme, e.g., but not limited to, a double-stranded stem, a single-stranded "catalytic core” sequence, a single-stranded loop or a single-stranded recognition sequence.
• These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such molecules can also act to cleave intramolecularly if that is desired.
• Such enzymatic molecules can be targeted to virtually any RNA transcript.
• Such molecules also include nucleic acid molecules having a 3' or 5' non- nucleotide, useful as a capping group to prevent exonuciease digestion.
• Non-nucleotide mimetics useful in this invention are generally described above and in Usman et al. WO 95/06731. Those in the art will recognize that these mimetics can be incorporated into an enzymatic molecule by standard techniques at any desired location. Suitable choices can be made by standard experiments to determine the best location, e.g., by synthesis of the molecule and testing of its enzymatic activity. The optimum molecule will contain the known ribonucleotides needed for enzymatic activity, and will have non-nucleotides which change the structure of the molecule in the least way possible. What is desired is that several nucleotides can be substituted by one non-nucleotide to save synthetic steps in enzymatic molecule synthesis and to provide enhanced stability of the molecule compared to RNA or even DNA.
• This invention relates to the synthesis, deprotection, and purification of enzymatic RNA or modified enzymatic RNA molecules in milligram to kilogram quantities with high biological activity. Such syntheses are generally detailed in Stinchcomb t al., WO 95/23225. This invention relates to the synthesis, deprotection, and purification of enzymatic RNA or modified enzymatic RNA molecules in milligram to kilogram quantities with high biological activity.
• RNA is synthesized and purified by methodologies based on: tetrazole to activate the RNA amidite, NH4OH to remove the exocyclic amino protecting groups, tetra-n-butylammonium fluoride (TBAF) to remove the 2'-OH alkylsilyl protecting groups, and gel purification and analysis of the deprotected RNA.
• TBAF tetra-n-butylammonium fluoride
• gel purification and analysis of the deprotected RNA In particular this applies to, but is not limited to, a certain class of RNA molecules, ribozymes. These may be formed either chemically or using enzymatic methods. Examples of the chemical synthesis, deprotection, purification and analysis procedures are provided by Usman et al., 1987 J. American Chem.
• the aforementioned chemical synthesis, deprotection, purification and analysis procedures are time consuming (10-15 m coupling times) and may also be affected by inefficient activation of the RNA amidites by tetrazole, time consuming (6-24 h) and incomplete deprotection of the exocyclic amino protecting groups by NH4OH, time consuming (6-24 h), incomplete and difficult to desalt TBAF-catalyzed removal of the alkylsilyl protecting groups, time consuming and low capacity purification of the RNA by gel electrophoresis, and low resolution analysis of the RNA by gel electrophoresis.
• Draper et al., WO 93/23569 (PCT/US93/04020) describes synthesis of ribozymes in two parts in order to aid in the synthetic process (see, ⁇ _&, p. 40).
• enzymatic nucleic acid molecules having non-nucleotides within their structure. Such non-nucleotides can be used in place of nucleotides to allow formation of an enzymatic nucleic acid.
• This invention relates to improved methods for synthesis of enzymatic nucleic acids and, in particular, hammerhead and hairpin motif ribozymes.
• This invention is advantageous over iterative chemical synthesis of ribozymes since the yield of the final ribozyme can be significantly increased.
• two partial ribozyme portions e.g.. a 20mer and a 17mer, can be synthesized in significantly higher yield, and the two reacted together to form the desired enzymatic nucleic acid.
• each n or n' is independently any desired nucleotide or non- nucleotide
• each filled-in circle represents pairing between bases or other entities
• the solid line represents a covalent bond.
• each n and n' may be a ribonucleotide, a 2'-methoxy-substituted nucleotide, or any other type of nucleotide which does not significantly affect the desired enzymatic activity of the final product (see Usman et al., supra).
• ribonucleotides are provided at rG5, rA6, rG8, rG12, and rA15.1.
• U4 and U7 may be abasic (i.e.. lacking the uridine moiety) or may be ribonucleotides, 2'- methoxy substituted nucleotides, or other such nucleotides.
• a9, a13, and a14 are preferably 2'-methoxy or may have other substituents.
• the synthesis of this hammerhead ribozyme is performed by synthesizing a 3' and a 5' portion as shown in a lower part of Fig. 68.
• Each 5' and 3' portion has a chemically reactive group X and Y, respectively.
• Non-limiting examples of such chemically reactive groups are provided in Fig. 69. These groups undergo chemical reactions to provide the bonds shown in Fig. 69.
• the X and Y can be used, in various combinations, in this invention to form a chemical linkage between two ribozyme portions.
• the invention features a method for synthesis of an enzymatically active nucleic acid (as defined by Draper, supra) by providing a 3' and a 5' portion of that nucleic acid, each having independently chemically reactive groups at the 5' and 3' positions, respectively.
• the reaction is performed under conditions in which a covalent bond is formed between the 3' and 5' portions by those chemically reactive groups.
• the bond formed can be, but is not limited to, either a disulfide, morpholino, amide, ether, thioether, amine, a double bond, a sulfonamide, carbonate, hydrazone or ester bond.
• the bond is not the natural bond formed between a 5' phosphate group and a 3' hydroxyl group which is made during normal synthesis of an oligonucleotide.
• more than two portions can be linked together using pairs of X and Y groups which allow proper formation of the ribozyme (see Figure 69).
• chemically reactive group is simply meant a group which can react with another group to form the desired bonds. These bonds may be formed under any conditions which will not significantly affect the structure of the resulting enzymatic nucleic acid. Those in the art will recognize that suitable protecting groups can be provided on the ribozyme portions.
• the nucleic acid has a hammerhead motif and the 3' and 5' portions each have chemically reactive groups in or immediately adjacent to the stem II region (see Fig. 1).
• the stem II region is evident in Fig. 1 between the bases termed a9 and rG12.
• the C and G within this stem defines the end of the stem II region.
• any of the n or n' moieties within the stem II region can be provided with a chemically reactive group.
• the chemically reactive groups need not be provided in the solid line portion but can be provided at any of the n or n'. In this way the length of each of the 5' and 3' portions can vary by several bases ( Figure 70).
• the chemically reactive group can be, but is not limited to, (CH 2 )nSH; (CH 2 )nNHR; (CH 2 )nX; ribose; COOH; (CH 2 )nPPh 3 ; (CH 2 )nS0 2 CI; (CH 2 )nCOR; (CH 2 ) n RNH or (CH 2 ) n OH, where, CH can be replaced by another group which forms a linking chain (which does not interfere with the terminal chemically reactive group) containing various atoms including, but not limited to CH 2 , such as methylenes, ether, ethylene glycol, thioethers, double bonds, aromatic groups and others, generally at most 20 such atoms are provided in the linking chain, most preferably only 5 - 10 atoms, and even more preferably only 3- 5 atoms; each n independently is an integer from 0 to 10 inclusive and may be the same or different; each R independently is a proton or an alkyl, alken
• the conditions include provision of Nal ⁇ 4 ' n contact with the ribose, and subsequent provision of a reducing group such as NaBH4 or NaCNB ⁇ ; or the conditions include provision of a coupling reagent.
• the invention features a mixture of the 5' and
• the hairpin ribozyme may be synthesized by inclusion of chemically reactive groups in helix IV and in other helices which are not critical to the enzymatic activity of the nucleic acid.
• RNA polymerase III (pol III) promoter is one found in DNA encoding 5S, U6, adenovirus VA1 , Vault, telomerase RNA, tRNA genes, etc., and is transcribed by RNA polymerase III (for a review see Geiduschek and Tocchini- Valentini, 1988 Annu. Rev. Biochem. 57, 873-914; Willis, 1993 Eur. J. Biochem. 212, 1-11).
• Type 1 pol III promoter consists of three cis-acting sequence elements downstream of the transcriptional start site a) 5'sequence element (A block); b) an intermediate sequence element (I block); c) 3' sequence element (C block). 5S ribosomal RNA genes are transcribed using the type 1 pol III promoter (Specht et al., 1991 Nucleic Acids Res. 19, 2189-2191.
• the type 2 pol III promoter is characterized by the presence of two cis- acting sequence elements downstream of the transcription start site. All Transfer RNA (tRNA), adenovirus VA RNA and Vault RNA (Kikhoefer et al., 1993, J. Biol. Chem. 268, 7868-7873) genes are transcribed using this promoter (Geiduschek and Tocchini-Valentini, 1988 supra; Willis, 1993 supra). The sequence composition and orientation of the two cis-acting sequence elements- A box (5' sequence element) and B box (3' sequence element) are essential for optimal transcription by RNA polymerase III.
• the type 3 pol III promoter contains all of the cis-acting promoter elements upstream of the transcription start site.
• Upstream sequence elements include a traditional TATA box (Mattaj et al., 1988 Cell 55, 435-442), proximal sequence element (PSE) and a distal sequence element (DSE; Gupta and Reddy, 1991 Nucleic Acids Res. 19, 2073-2075).
• PSE proximal sequence element
• DSE distal sequence element
• Examples of genes under the control of the type 3 pol III promoter are U6 small nuclear RNA (U6 snRNA) and Telomerase RNA genes.
• Epstein-Barr-virus-encoded RNAs EBER
• Xenopus seleno-cysteine tRNA human 7SL RNA
• EBER genes contain a functional A and B box (similar to type 2 pol III promoter). In addition they also require an EBER-specific TATA box and binding sites for ATF transcription factors (Howe and Shu, 1989 Cell 57,825-834).
• the seleno-cysteine tRNA gene contains a TATA box, PSE and DSE (similar to type 3 pol III promoter). Unlike most tRNA genes, the seleno- cysteine tRNA gene lacks a functional A box sequence element. It does require a functional B box (Lee et al., 1989 J. Biol. Chem. 264, 9696-9702).
• the human 7SL RNA gene contains an unique sequence element downstream of the transcriptional start site. Additionally, upstream of the transcriptional start site, the 7SL gene contains binding sites for ATF class of transcription factors and a DSE (Bredow et al., 1989 Gene 86, 217-225).
• Gilboa WO 89/11539 and Gilboa and Sullenger WO 90/13641 describe transformation of eucaryotic cells with DNA under the control of a pol III promoter. They state:
• pol III promoters In an attempt to improve antisense RNA synthesis using stable gene transfer protocols, the use of pol III promoters to drive the expression of antisense RNA can be considered.
• the underlying rationale for the use of pol III promoters is that they can generate substantially higher levels of RNA transcripts in cells as compared to pol II promoters. For example, it is estimated that in a eucaryotic cell there are about 6 x 10 7 1- RNA molecules and 7 x 10 5 mRNA molecules, i.e., about 100 fold more pol III transcripts of this class than total pol II transcripts. Since there are about 100 active t-RNA genes per cell, each t-RNA gene will generate on the average RNA transcripts equal in number to total pol II transcripts.
• a t-RNA (pol III) based transcriptional unit may be able to generate 100 fold to 10,000 fold more RNA than a pol II based transcriptional unit.
• Sisodia have fused the Adenovirus VA-1 promoter to various DNA sequences (the herpes TK gene, globin and tubulin) and used transfection protocols to transfer the resulting DNA constructs into cultured cells which resulted in transient synthesis of RNA in the transduced cell.
• De la Pena and Zasloff have expressed a t-RNA-Herpes TK fusion DNA construct upon microinjection into frog oocytes.
• Jennings and Molloy have constructed an antisense RNA template by fusing the VA-1 gene promoter to a DNA fragment derived from SV40 based vector which also resulted in transient expression of antisense RNA and limited inhibition of the target gene".
• RNA transcript [Citations omitted.]
• the authors describe a fusion product of a chimeric tRNA and an RNA product (see Fig. 1C of WO 90/13641).
• a human tRNA metj derivative 3-5 was derived from a cloned human tRNA gene by deleting 19 nucleotides from the 3" end of the gene.
• the authors indicate that the truncated gene can be transcribed if a termination signal is provided, but that no processing of the 3 1 end of the RNA transcript takes place.
• Adeniyi-Jones et al.,1984 Nucleic Acids Res. 12, 1101-1115 describe certain constructions which "may serve as the basis for utilizing the tRNA gene as a 'portable promoter' in engineered genetic constructions.”
• the authors describe the production of a so-called ⁇ 3'-5 in which 11 nucleotides of the 3'- end of the mature tRNAj met sequence are replaced by a plasmid sequence, and are not processed to generate a mature tRNA.
• tRNAj met 3' deletion plasmids described in this study suggest their potential use in certain engineered genetic constructions.
• the tRNA gene could be used to promote transcription of theoretically any DNA sequence fused to the 3' border of the gene, generating a fusion gene which would utilize the efficient polymerase III promoter of the human tRNAj me * gene.
• a tRNAj m ⁇ t deletion mutant such as ⁇ 3'-4, a long read-through transcript would be generated in vivo (dependent, of course, on the absence of effective RNA polymerase III termination sequences).
• Fusion of the DNA sequence to a tRNAj met deletion mutant such as ⁇ 3'-5 would lead to the generation of a co-transcript from which subsequent processing of the tRNA leader at the 5' portion of the fused transcript would be blocked.
• Control over processing may be of some biological use in engineered constructions, as suggested by properties of mRNA species bearing tRNA sequences as 5' leaders in prokaryotes.
• Such "dual transcripts" code for several predominant bacterial proteins such as EF-Tu and may use the tRNA leaders as a means of stabilizing the transcript from degradation in vivo.
• the potential use of the tRNAj met gene as a "promoter leader" in eukaryotic systems has been realized recently in our laboratory.
• Fusion genes consisting of the deleted tRNAj met sequences contained on plasmids ⁇ 3'-4 and ⁇ 3'-5 in front of a promoter-less Herpes simplex type I thymidine kinase gene yield viral-specific enzyme resulting from RNA polymerase III dependent transcription in both X. laevis oocytes and somatic cells". [References omitted]. Sullenger et al., 1990 Cell 63, 601-619, describe over-expression of Tflft-containing sequences using a chimeric tRNAj-T- S t-Ty-lf? transcription unit in a double copy (DC) murine retroviral vector.
• DC double copy
• RNA pol Ill-based transcription system to stably express high levels of foreign RNA sequences in cells may have other important applications. Foremost, it may significantly improve the ability to inhibit endogenous genes in eucaryotic cells for the study of gene expression and function, whether antisense RNA, ribozymes, or competitors of sequence-specific binding factors are used.
• tRNA-driven transcription systems may be particularly useful for introducing "mutations" into the germ line, i.e., for generating transgenic animals or transgenic plants. Since tRNA genes are ubiquitously expressed in all cell types, the chimeric tRNA genes may be properly expressed in all tissues of the animal, in contrast to the more idiosyncratic behavior of RNA pol ll-based transcription units. However, homologous recombination represents a more elegant although, at present, very cumbersome approach for introducing mutations into the germ line. In either case, the ability to generate transgenic animals or plants carrying defined mutations will be an extremely valuable experimental tool for studying gene function in a developmental context and for generating animal models for human genetic disorders. In addition, tRNA-driven gene inhibition strategies may also be useful in creating pathogen- resistant livestock and plants. [References omitted.]
• VAI promoter to express a hairpin ribozyme.
• the resulting transcript consisted of the first 104 nucleotides of the VAI RNA, followed by the ribozyme sequence and the terminator sequence.
• Pol Ill-based vectors are described in Stinchcomb et al., WO 95/23225.
• ribozyme motifs By engineering ribozyme motifs applicant has designed several ribozymes directed against stromelysin mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance.
• the ribozymes are tested for function in vivo by analyzing stromelysin expression levels. Ribozymes are delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, and/or by expression from DNA/RNA vectors. Stromelysin expression is monitored by biological assays, ELISA, by indirect immunofluoresence, and/or by FACS analysis. Stromelysin mRNA levels are assessed by Northern analysis, RNAse protection, primer extension analysis and/or quantitative RT-PCR. Ribozymes that block the induction of stromelysin activity and/or stromelysin mRNA by more than 50% are identified.
• Ribozymes targeting selected regions of mRNA associated with arthritic disease are chosen to cleave the target RNA in a manner which preferably inhibits translation of the RNA. Genes are selected such that inhibition of translation will preferably inhibit cell replication, e.g.. by inhibiting production of a necessary protein or prevent production of an undesired protein, e.g.. stromelysin. Selection of effective target sites within these critical regions of mRNA may entail testing the accessibility of the target RNA to hybridization with various oligonucleotide probes. These studies can be performed using RNA or DNA probes and assaying accessibility by cleaving the hybrid molecule with RNaseH (see below).
• ribozyme probes designed from secondary structure predictions of the mRNAs, and assaying cleavage products by polyacrylamide gel electrophoresis (PAGE), to detect the presence of cleaved and uncleaved molecules.
• PAGE polyacrylamide gel electrophoresis
• ribozyme target sites within the rabbit stromelysin mRNA sequence (1795 nucleotides) were located and aligned with the human target sites. Because the rabbit stromelysin mRNA sequence has an 84% sequence identity with the human sequence, many ribozyme target sites are also homologous. Thus, the rabbit has potential as an appropriate animal model in which to test ribozymes that are targeted to human stromelysin but have homologous or nearly homologous cleavage sites on rabbit stromelysin mRNA as well (Tables AII-AVI, AVIII & AIX ).
• 316 UH sites in the rabbit sequence are identical with the corresponding site in the human sequence with respect to at least 14 nucleotides surrounding the potential ribozyme cleavage sites.
• the nucleotide in the RNA substrate that is immediately adjacent (5") to the cleavage site is unpaired in the ribozyme- substrate complex (see Fig. 1 ) and is consequently not included in the comparison of human and rabbit potential ribozyme sites.
• human ribozyme target sites for continued testing the presence of identical or nearly identical sites in the rabbit sequence is considered.
• ribozyme target sites were subjected to further analysis using computer folding programs (Mulfold or a Macintosh-based version of the following program, LRNA (Zucker (1989) Science 244:48). to determine if 1) the target site is substantially single-stranded and therefore predicted to be available for interaction with a ribozyme, 2) if a ribozyme designed to that site is predicted to form stem II but is generally devoid of any other intramolecular base pairing, and 3) if the potential ribozyme and the sequence flanking both sides of the cleavage site together are predicted to interact correctly.
• computer folding programs Mefold or a Macintosh-based version of the following program, LRNA (Zucker (1989) Science 244:48).
• the sequence of Stem II can be altered to maintain a stem at that position but minimize intramolecular basepairing with the ribozyme's substrate binding arms. Based on these minimal criteria, and including all the sites that are identical in human and rabbit stromelysin mRNA sequence, a subset of 66 potential superior ribozyme target sites was chosen (as first round targets) for continued analysis. These are SEQ. ID.
• NOS. 34, 35, 37, 47, 54, 57, 61 , 63, 64, 66, 76, 77, 79, 87, 88, 96, 97, 98, 99, 100, 107, 110, 121 , 126, 128, 129, 133, 140, 146, 148, 151 , 162, 170, 179, 188, 192, 194, 196, 199, 202, 203, 207, 208, 218, 220, 223, 224, 225, 227, 230, 232, 236, 240, 245, 246, 256, 259, 260, 269, 280, 281 , 290, 302, 328, 335 and 353 (see Table AIM).
• RNAse H assay is carried out. Using this assay, the accessibility of a potential ribozyme target site to a DNA oligonucleotide probe can be assessed without having to synthesize a ribozyme to that particular site. If the complementary DNA oligonucleotide is able to hybridize to the potential ribozyme target site then RNAse H, which has the ability to cleave the RNA of a DNA/RNA hybrid, will be able to cleave the target RNA at that particular site.
• RNAse H Specific cleavage of the target RNA by RNAse H is an indication that that site is "open” or “accessible” to oligonucleotide binding and thus predicts that the site will also be open for ribozyme binding.
• RNAse H By comparing the relative amount of specific RNAse H cleavage products that are generated for each DNA oligonucleotide/site, potential ribozyme sites can be ranked according to accessibility.
• DNA oligonucleotides (generally 13-15 nucleotides in length) that are complementary to the potential target sites are synthesized.
• Body-labeled substrate RNAs (either full-length RNAs or -500-600 nucleotide subfragments of the entire RNA) are prepared by in vitro transcription in the presence of a ⁇ 2 P-labeled nucleotide. Unincorporated nucleotides are removed from the 32p.
• the 32 P-labeled substrate RNA is pre-incubated with the specific DNA oligonucleotide (1 ⁇ M and 0.1 ⁇ M final concentration) in 20 mM Tris-HCl, pH 7.9, 100 mM KCI, 10 mM MgCI 2 , 0.1 mM EDTA, 0.1 mM DTT at 37°C for 5 minutes. An excess of RNAse H (0.8 units/10 ⁇ l reaction) is added and the incubation is continued for 10 minutes.
• RNAse H-cleaved RNA products are separated from uncleaved RNA on denaturing polyacrylamide gels, visualized by autoradiography and the amount of cleavage product is quantified.
• RNAse H analysis on the 66 potential ribozyme sites was carried out and those DNA oligonucleotides/sites that supported the most RNAse H cleavage were determined. These assays were carried out using full-length human and rabbit stromelysin RNA as substrates. Results determined on human stromelysin RNA indicated that 23 of the 66 sites supported a high level of RNAse H cleavage, and an additional 13 supported a moderate level of RNAse H cleavage. Twenty-two sites were chosen from among these two groups for continued study.
• RNAse H accessibility on rabbit stromelysin RNA was determined, but was not used as a specific criteria for these choices.
• Those DNA oligonucleotides that are not totally complementary to the rabbit sequence may not be good indicators of the relative amount of RNAse H cleavage, possibly because the mismatch leads to less efficient hybridization of the DNA oligonucleotide to the mismatched RNA substrate and therefore less RNAse H cleavage is seen.
• Example 4 Analysis of Ribozymes Ribozymes were then synthesized to 22 sites (Table AV) predicted to be accessible as judged the RNAse H assay. Eleven of these 22 sites are identical to the corresponding rabbit sites. The 22 sites are SEQ. ID, NOS.: 34, 35, 57, 125, 126, 127, 128, 129, 140, 162, 170, 179, 188, 223, 224, 236, 245, 246, 256, 259, 260, 281.
• the 22 ribozymes were chemically synthesized with recognition arms of either 7 nucleotides or 8 nucleotides, depending on which ribozyme alone and ribozyme-substrate combinations were predicted by the computer folding program (Mulfold) to fold most correctly. After synthesis, ribozymes are either purified by HPLC or gel purified. These 22 ribozymes were then tested for their ability to cleave both human and rabbit full-length stromelysin RNA. Full-length, body-labeled stromelysin RNA is prepared by in vitro transcription in the presence of [ ⁇ - 3 2 P]CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
• Assays are performed by prewarming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37°C, 10 mM MgCI ) and the cleavage reaction is initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that has also been prewarmed in cleavage buffer.
• substrate RNA maximum of 1-5 nM
• assays are carried out for 1 hour at 37°C using a final concentration of 1 ⁇ M and 0.1 ⁇ M ribozyme, e, . , ribozyme excess.
• the reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol FF after which the sample is heated to 95°C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Full-length substrate RNA and the specific RNA products generated by ribozyme cleavage are visualized on an autoradiograph of the gel.
• RNA cleavage products of the appropriate lengths were visualized.
• the size of the RNA was judged by comparison to molecular weight standards electrophoresed in adjacent lanes of the gel.
• the fraction of substrate RNA cleaved during a ribozyme reaction can be used as an assessment of the activity of that ribozyme in vitro.
• the activity of these 22 ribozymes on full-length substrate RNA ranged from approximately 10% to greater than 95% of the substrate RNA cleaved in the ribozyme cleavage assay using 1 ⁇ M ribozyme as described above.
• ribozymes to these seven sites were designed that had alterations in the binding arm lengths.
• a complete set of ribozymes was synthesized that included ribozymes with binding arms of 6 nucleotides, 7 nucleotides, 8 nucleotides, 10 nucleotides and 12 nucleotides, ej., 5 ribozymes to each site.
• These ribozymes were gel- purified after synthesis and tested in ribozyme cleavage assays as described above.
• Example 6 Testing the efficacy of ribozymes in cell culture
• ribozymes with 7 nucleotide arms were synthesized with 2' O- methyl modifications on the 5 nucleotides at the 5' end of the molecule and on the 5 nucleotides at the 3' end of the molecule.
• ribozymes to the same sites but with 12 nucleotide arms were also synthesized with the 2' O methyl modifications at the 5 positions at the end of both binding arms.
• Inactive ribozymes that contain 2 nucleotide changes in the catalytic core region were also prepared for use as controls.
• the catalytic core in the inactive ribozymes i s C U LA U G A G G C C G A A A G G C C G A L ve rs u s CUG.AUGAGGCCGAAAGGCCGAA in the active ribozymes.
• the inactive ribozymes show no cleavage activity in vitro when measured on full-length RNA in the typical ribozyme cleavage assay at a 1 ⁇ M concentration for 1 hour.
• the general assay was as follows: Fibroblasts, which produce stromelysin, are serum-starved overnight and ribozymes or controls are offered to the cells the next day. Cells are maintained in serum-free media.
• the ribozyme can be applied to the cells as free ribozyme, or in association with various delivery vehicles such as cationic lipids (including TransfectamTM, LipofectinTM and LipofectamineTM), conventional liposomes, non-phospholipid liposomes or biodegradable polymers.
• lnterleukin-1 ⁇ typically 20 units/ml
• stromelysin can then be monitored over a time course, usually up to 24 hours.
• a ribozyme is effective in cleaving stromelysin mRNA within a cell, the amount of stromelysin mRNA will be decreased or eliminated.
• a decrease in the level of cellular stromelysin mRNA, as well as the appearance of the RNA products generated by ribozyme cleavage of the full-length stromelysin mRNA, can be analyzed by methods such as Northern blot analysis, RNAse protection assays and/or primer extension assays.
• the effect of ribozyme cleavage of cellular stromelysin mRNA on the production of the stromelysin protein can also be measured by a number of assays.
• stromelysin secreted into the media of lnterleukin-1 ⁇ -induced human synovial fibroblasts was measured by ELISA using an antibody that recognizes human stromelysin.
• a TransfectamTM-ribozyme complex (0.15 ⁇ M ribozyme final concentration) was offered to 2-4 x 10 5 serum-starved cells for 3 hours prior to induction with lnterleukin-1 ⁇ .
• the TransfectamTM was prepared according to the manufacturer (Promega Corp.) except that 1 :1 (w/w) dioleoyi phosphatidylethanolamine was included.
• the TransfectamTM-ribozyme complex was prepared in a 5:1 charge ratio.
• the control is TransfectamTM alone applied to the cell.
• Inactive ribozymes with 7 nucleotide arms or 12 nucleotide arms have the two inactivating changes to the catalytic core that are described above.
• Cell samples were prepared in duplicate and the assay was carried out on several dilutions of the conditioned media from each sample. Results of the ELISA are presented below as a percent of stromelysin present vs. the control (NO RZ) which is set at 100%.
• RZ 617H 7/7 and RZ 820H 7/7 had better cleavage activity on full-length RNA substrates than ribozymes with 12 nucleotide arms directed to the same sites (617H 12/12 and RZ 820H 12/12).
• stromelysin detection is to visualize stromelysin protein in the cells by immunofluorescence.
• cells are treated with monensin to prevent protein secretion from the cell.
• the stromelysin retained by the cells after monensin addition can then be visualized by immunofluorescence using either conventional or confocal microscopy.
• cells were serum-starved overnight and treated with ribozyme the following day for several hours.
• Monensin was then added and after -5-6 hours, monensin-treated cells were fixed and permeabilized by standard methods and incubated with an antibody recognizing human stromelysin.
• Rabbit synovial fibroblasts were also treated with RZ 617H 7/7 or RZ 820H 7/7, as well as with the two corresponding ribozymes (RZ 617R 7/7 or RZ 820R 7/7) that each have the appropriate one nucleotide change to make them completely complementary to the rabbit target sequence.
• Relative to controls that had no ribozyme treatment immunofluorescence in Interleukin- 1 ⁇ -induced rabbit synovial fibroblasts was visibly decreased by treatment with these four ribozymes, whether specific for rabbit or human mRNA sequence.
• the antibody to human stromelysin was used for the immunofluorescence study in rabbit synovial fibroblasts.
• the primer extension assay was used to detect full-length RNA as well as the 3' ribozyme cleavage products of the RNA of interest.
• the method involves synthesizing a DNA primer (generally -20 nucleotides in length) that can hybridize to a position on the RNA that is downstream (3') of the putative ribozyme cleavage site. Before use, the primer was labeled at the 5' end with 32 P[ATP] using T4 polynucleotide kinase and purified from a gel. The labeled primer was then incubated with a population of nucleic acid isolated from a cellular lysate by standard procedures.
• the reaction buffer was 50 mM Tris- HCI, pH 8.3, 3 mM MgCI 2 , 20 mM KCI, and 10 mM DTT.
• a 30 minute extension reaction follows, in which all DNA primers that have hybridized to the RNA were substrates for reverse transcriptase, an enzyme that will add nucleotides to the 3' end of the DNA primer using the RNA as a template.
• Reverse transcriptase was obtained from Life Technologies and is used essentially as suggested by the manufacturer. Optimally, reverse transcriptase will extend the DNA primer, forming cDNA, until the end of the RNA substrate is reached.
• the cDNA product will be shorter than the resulting cDNA product of a full-length, or uncleaved RNA substrate.
• the differences in size of the 32 P-labeled cDNAs produced by extension can then be discriminated by electrophoresis on a denaturing polyacrylamide gel and visualized by autoradiography.
• RNA substrate Strong secondary structure in the RNA substrate can, however, lead to premature stops by reverse transcriptase. This background of shorter cDNAs is generally not a problem unless one of these prematurely terminated products electrophoreses in the expected position of the ribozyme-cleavage product of interest. Thus, 3' cleavage products are easily identified based on their expected size and their absence from control lanes. Strong stops due to secondary structure in the RNA do, however, cause problems in trying to quantify the total full-length and cleaved RNA present. For this reason, only the relative amount of cleavage can easily be determined.
• the primer extension assay was carried out on RNA isolated from cells that had been treated with TransfectamTM-complexed RZ 617H 7/7, RZ 820H 7/7, RZ 617H 12/12 and RZ 820H 12/12. Control cells had been treated with TransfectamTM alone. Primer extensions on RNA from cells treated with the TransfectamTM complexes of the inactive versions of these four ribozymes were also prepared.
• the 20 nucleotide primer sequence is 5' AATGAAAACGAGGTCCTTGC 3' and it is complementary to a region about 285 nucleotides downstream of ribozyme site 820.
• the cDNA length for the 3' cleavage product is 488 nucleotides, for 820 the cDNA product is 285 nucleotides.
• Full-length cDNA will be 1105 nucleotides in length.
• 1 ml of 0.15 ⁇ M ribozyme was offered to -2-3 x 10 ⁇ serum-starved human synovial fibroblasts. After 3 hours, 20 units/ml lnterleukin-1 ⁇ was added to the cells and the incubation continued for 24 hours.
• 3 P-labeled cDNAs of the correct sizes for the 3' products were clearly visible in lanes that contained RNA from cells that had been treated with active ribozymes to sites 617 and 820. Ribozymes with 7 nucleotide arms were judged to be more active than ribozymes with 12 nucleotide arms by comparison of the relative amount of 3' cleavage product visible. This correlates well with the data obtained by ELISA analysis of the conditioned media from these same samples. In addition, no cDNAs corresponding to the 3' cleavage products were visible following treatment of the cells with any of the inactive ribozymes.
• RNA substrate was not occurring during the preparation of the cellular RNA or during the primer extension reaction itself.
• One control was to add body-labeled stromelysin RNA, prepared by in vitro transcription, to the cellular lysate. This lysate was then subjected to the typical RNA preparation and primer extension analysis except that non-radioactive primer was used. If ribozymes that are present in the cell at the time of cell lysis are active under any of the conditions during the subsequent analysis, the added, body-labeled stromelysin RNA will become cleaved. This, however, is not the case.
• RNAse protection assay is carried out essentially as described in the protocol provided with the Lysate Ribonuclease Protection Kit (United States Biochemical Corp.)
• the probe for RNAse protection is an RNA that is complementary to the sequence surrounding the ribozyme cleavage site.
• This "antisense" probe RNA is transcribed in vitro from a template prepared by the polymerase chain reaction in which the 5' primer was a DNA oligonucleotide containing the T7 promoter sequence.
• the probe RNA is body labeled during transcription by including 3 P[CTP] in the reaction and purified away from unincorporated nucleotide triphosphates by chromatography on G-50 Sephadex.
• the probe RNA (100,000 to 250,000 cpms) is allowed to hybridize overnight at 37°C to the RNA from a cellular lysate or to RNA purified from a cell lysate.
• RNAse T- j and RNAse A are added to degrade all single-stranded RNA and the resulting products are analyzed by gel electrophoresis and autoradiography. By this analysis, full-length, uncleaved target RNA will protect the full-length probe.
• RNAse digestion For ribozyme-cleaved target RNAs, only a portion of the probe will be protected from RNAse digestion because the cleavage event has occurred in the region to which the probe binds. This results in two protected probe fragments whose size reflects the position at which ribozyme cleavage occurs and whose sizes add up to the size of the full-length protected probe.
• RNAse protection analysis was carried out on cellular RNA isolated from rabbit synovial fibroblasts that had been treated either with active or inactive ribozyme.
• the ribozymes tested had 7 nucleotide arms specific to the rabbit sequence but corresponding to human ribozyme sites 617 and 820 (i.e. RZ 617R 7/7, RZ 820R 7/7).
• the inactive ribozymes to the same sites also had 7 nucleotide arms and included the two inactivating changes described above.
• the inactive ribozymes were not active on full-length rabbit stromelysin RNA in a typical 1 hour ribozyme cleavage reaction in vitro at a concentration of 1 ⁇ M.
• ribozyme For all samples, one ml of 0.15 ⁇ M ribozyme was administered as a TransfectamTM complex to serum-starved cells. Addition of Interleukin-l ⁇ followed 3 hours later and cells were harvested after 24 hours. For samples from cells treated with either active ribozyme tested, the appropriately-sized probe fragments representing ribozyme cleavage products were visible. For site 617, two fragments corresponding to 125 and 297 nucleotides were present, for site 820 the two fragments were 328 and 94 nucleotides in length. No protected probe fragments representing RNA cleavage products were visible in RNA samples from cells that not been treated with any ribozyme, or in cells that had received the inactive ribozymes. Full-length protected probe (422 nucleotides in length) was however visible, indicating the presence of full-length, uncleaved stromelysin RNA in these samples.
• Ribozymes can be delivered to fibroblasts complexed to a cationic lipid or in free form.
• an appropriate dilution of stock ribozyme (final concentration is usually 1.5 ⁇ M) is made in serum-free medium; if a radioactive tracer is to be used ( e ⁇ ., 3 P), the specific activity of the ribozyme is adjusted to 800-1200 cpm/pmol.
• the lipid is first prepared as a stock solution containing 1/1 (w/w) dioleoylphosphatidylcholine (DOPE).
• DOPE dioleoylphosphatidylcholine
• Ribozyme is mixed with the Transfectam/DOPE mixture at a 1/5 (RZ/TF) charge ratio; for a 36-mer ribozyme, this is a 45-fold molar excess of Transfectam (Transfectam has 4 positive charges per molecule). After a 10 min incubation at room temperature, the mixture is diluted and applied to cells, generally at a ribozyme concentration of 0.15 ⁇ M. For 3 P experiments, the specific activity of the ribozyme is the same as for the free ribozyme experiments.
• Transfectam-ribozyme cpm's are cell-associated (in a nuclease-resistant manner). Of this, about 10-15% of the cpm's represent intact ribozyme; this is about 20-25 million ribozymes per cell. For the free ribozyme, about 0.6% of the offered dose is cell-associated after 24 hours. Of this, about 10-15% is intact; this is about 0.6-0.8 million ribozymes per cell.
• Example 11 //? vitro cleavage of stromelysin mRNA bv HH ribozvmes
• ribozymes targeted against some of the sites listed in example 2 and Table 3, were synthesized. These ribozymes were extensively modified such that: 5' terminal nucleotides contain phosphorothioate substitutions; except for five ribose residues in the catalytic core, all the other 2'-hydroxyl groups within the ribozyme were substituted with either 2'-0-methyl groups or 2'-C-allyl modifications.
• the aforementioned modifications are meant to be non-limiting modifications. Those skilled in the art will recognize that other embodiments can be readily generated using the techniques known in the art.
• ribozymes were tested for their ability to cleave RNA substrates in vitro.
• RNA cleavage by HH ribozymes targeted to sites 21 , 463, 1049, 1366, 1403, 1410 and 1489 was assayed at 37°C.
• Substrate RNAs were 5' end-labeled using [ ⁇ - 32 P]ATP and T4 polynucleotide kinase enzyme.
• RNA and 40 nM ribozyme were denatured separately by heating to 90°C for 2 min followed by snap cooling on ice for 10 min.
• the substrate and the ribozyme reaction mixtures were renatured in a buffer containing 50 mM Tris-HCl, pH 7.5 and 10 mM MgCI at 37°C for 10 min.
• Cleavage reaction was initiated by mixing the ribozyme and the substrate RNA and incubating at 37°C. Aliquots of 5 ⁇ l were taken at regular intervals of time and the reaction quenched by mixing with an equal volume of formamide stop mix. The samples were resolved on a 20% polyacrylamide/urea gel.
• FIG. 7 A plot of percent RNA substrate cleaved as a function of time is shown in Fig. 7. The plot shows that all six HH ribozymes cleaved the target RNA efficiently. Some HH ribozymes were, however, more efficient than others .e.g.. 1049HH cleaves faster than 1366HH).
• Ribozymes were assayed on either human foreskin fibroblasts(HS-27) cell line or primary human synovial fibroblasts (HSF). All cells were plated the day before the assay in media containing 10% fetal bovine serum in 24 well plates at a density of 5x10 4 cells/well. At 24 hours after plating, the media was removed from the wells and the monolayers were washed with Dulbeccos phosphate buffered saline (PBS). The cells were serum starved for 24 h by incubating the cells in media containing 0.5% fetal bovine serum (FBS; 1 ml/well).
• FBS fetal bovine serum
• Ribozyme/lipid complexes were prepared as follows: Ribozymes and LipofectAMINE were diluted separately in serum-free DMEM plus 20 mM Hepes pH 7.3 to 2X final concentration, then equal volumes were combined, vortexed and incubated at 37°C for 15 minutes. The charge ratio of LipofectAmine: ribozyme was 3:1. Cells were washed twice with PBS containing Ca 2+ and Mg + . Cells were then treated the ribozyme/lipid complexes and incubated at 37°C for 1.5 hours. FBS was then added to a final concentration of 10%.
• ribozyme containing solution was removed and 0.5 ml DMEM containing 50 u/ml IL-1 , 10% FBS, 20 mM Hepes pH 7.3 added. Supernatants were harvested 16 hours after IL-1 induction and assayed for stromelysin expression by ELISA. Polyclonal antibody against Matrix Metalloproteinase 3 (Biogenesis, NH) was used as the detecting antibody and anti-stromelysin monoclonal antibody was used as the capturing antibody in the sandwich ELISA (Maniatis et al., supra) to measure stromelysin expression.
• Matrix Metalloproteinase 3 Biogenesis, NH
• Example 12 Ribozyme-Mediated Inhibition of Stromelysin Expression in human fibroblast cells Referring to Figs. 8 through 13, HH ribozymes, targeted to sites 21 , 463,
• stromelysin protein levels were measured using a sensitive ELISA protocol as described above.
• + IL-1 in the figures mean that cells were treated with IL-1 to induce the expression of stromelysin expression.
• -IL-1 means that the cells were not treated.
• Figs. 8 through 13 show the dramatic reduction in the levels of stromelysin protein expressed in cells that were transfected with active HH ribozymes. This decrease in the level of stromelysin production is over and above some non-specific inhibition seen in cells that were transfected with catalytically inactive ribozymes.
• HH ribozyme (1049HH), targeted to site 1049 within human stromelysin-1 mRNA, for animal studies because site 1049 is 100% identical to site 1060 (Tables AIM and AVI) within rabbit stromelysin mRNA. This has enabled applicant to compare the efficacy of the same ribozyme in human as well as in rabbit systems.
• mice Male New Zealand White Rabbits (3-4 Kg) were anaesthetized with ketamine-HCI/xylazine and injected intra-articularly (IT.) in both knees with 100 ⁇ g ribozyme (e.g., SEQ. ID. NO. 202) in 0.5 ml phosphate buffered saline (PBS) or PBS alone (Controls).
• ribozyme e.g., SEQ. ID. NO. 202
• PBS phosphate buffered saline
• Controls Controls.
• the IL-1 human recombinant IL-1 ⁇ , 25 ng
• Each rabbit received IL-1 in one knee and PBS alone in the other.
• the synovium was harvested 6 hours post IL-1 infusion, snap frozen in liquid nitrogen, and stored at -80°C.
• RNA is extracted with TRIzol reagent (GIBCO BRL, Gaithersburg, MD), and was analyzed by Northern-blot analysis and/or RNase-protection assay. Briefly, 0.5 ⁇ g cellular RNA was separated on 1.0 % agarose/formaldehyde gel and transferred to Zeta-Probe GT nylon membrane (Bio-Rad, Hercules, CA) by capillary transfer for -16 hours. The blots were baked for two hours and then pre-hybridized for 2 hours at 65°C in 10 ml Church hybridization buffer (7 % SDS, 500 mM phosphate, 1 mM EDTA, 1% Bovine Serum Albumin).
• the blots were hybridized at 65°C for -16 hours with 10 6 cpm/ml of full length 3 2 P-labeled complementary RNA (cRNA) probes to rabbit stromelysin mRNA (cRNA added to the pre-hybridization buffer along with 100 ⁇ l 10mg/ml salmon sperm DNA).
• the blot was rinsed once with 5% SDS, 25 mM phosphate, 1 mM EDTA and 0.5% BSA for 10 min at room temperature. This was followed by two washes (10 min each wash) with the same buffer at 65°C, which was then followed by two washes (10 min each wash) at 65°C with 1% SDS, 25 mM phosphate and 1 mM EDTA.
• the blot was autoradiographed.
• the blot was reprobed with a 100 nt cRNA probe to 18S rRNA as described above.
• the stromelysin expression was quantified on a scanning densitometer, which is followed by normalization of the data to the 18S rRNA band intensities.
• catalytically active 1049HH ribozyme mediates a decrease in the expression of stromelysin expression in rabbit knees.
• the inhibition appears to be sequence-specific and ranges from 50-70%.
• Example 14 Phosphorothioate-substituted Ribozymes inhibit stromelysin expression in Rabbit Knee Ribozymes containing four phosphorothioate linkages at the 5' termini enhance ribozyme efficacy in mammalian cells.
• applicant has designed and synthesized hammerhead ribozymes targeted to site 1049 within stromelysin RNA, wherein, the ribozymes contain five phosphorothioate linkages at their 5' and 3' termini. Additionally, these ribozymes contain 2'-0- methyl substitutions at 30 nucleotide positions, 2'-C-allyl substitution at U4 position and 2'-OH at five positions (Fig 17A).
• ribozymes were administered to rabbit knees to test for ribozyme efficacy.
• applicant has also designed and synthesized hammerhead ribozymes targeted to three distinct sites within stromelysin RNA, wherein, the ribozymes contain four phosphorothioate linkages at their 5' termini.
• these ribozymes contain 2'-0-methyl substitutions at 29 nucleotide positions, 2'-amino substitutions at U4 and U7 positions and 2'-OH at five positions. As described above, these ribozymes were administered to rabbit knees to test for ribozyme efficacy. As shown in Figures 18-21 , ribozymes targeted to sites 1049, 1363 and 1366 are all efficacious in rabbit knee. All three ribozymes decreased the level of stromelysin RNA in rabbit knee by about 50 %.
• B7-1 , B7-2, B7-3 and CD40 are attractive ribozyme targets by several criteria.
• the molecular mechanism of T cell activation is well-established. Efficacy can be tested in well-defined and predictive animal models. The clinical end-point of graft rejection is clear. Since delivery would be ex vivo, treatment of the correct cell population would be assured. Finally, the disease condition is serious and current therapies are inadequate. Whereas protein- based therapies would induce anergy against all antigens encountered during the several week treatment period, ex vivo ribozyme therapy provides a direct and elegant approach to truly donor-specific anergy. Similarly, autoimmune diseases and allergies can be prevented or treated by reversing the devastating course of immune response to self- antigens. Specifically, nucleic acids of this inventions can dampen the response to naturally occuring antigens.
• Example 15 B7-1. B7-2. B7-3 and/or CD40 Hammerhead ribozvmes
• ribozyme motifs By engineering ribozyme motifs we have designed several ribozymes directed against B7-1 , B7-2, B7-3 and/or CD40 encoded mRNA sequences.
• ribozymes were synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave target sequences in vitro was evaluated.
• B7-1 , B7-2, B7-3 and/or CD40 can be induced to express endogenous B7-1 , B7-2, B7-3 and/or CD40 .
• murine splenic cells can be isolated and induced, to express B7-1 or B7-2, with IL-4 or recombinant CD40 ligand.
• B7-1 and B7-2 can be detected easily with monoclonal antibodies.
• Use of appropriate flourescent reagents and flourescence-activated cell-sorting (FACS) will permit direct quantitation of surface B7-1 and B7-2 on a cell-by-cell basis.
• Active ribozymes are expected to directly reduce B7-1 or B7-2 expression. Ribozymes targeted to CD40 would prevent induction of B7-2 by CD40 ligand.
• B7-1 , B7-2, B7-3 and/or CD40 protein levels can be measured clinically or experimentally by FACS analysis.
• B7-1, B7-2, B7-3 and/or CD40 encoded mRNA levels will be assessed by Northern analysis, RNase-protection, primer extension analysis and/or quantitative RT-PCR.
• Ribozymes that block the induction of B7-1, B7- 2, B7-3 and/or CD40 activity and/or B7-1 , B7-2, B7-3 and/or CD40 protein encoding mRNAs by more than 20% in vitro will be identified.
• EAE allergic encephalomyelitis
• RNA ribozymes and/or genes encoding them will be delivered by either free delivery, liposome delivery, cationic lipid delivery, adeno-associated virus vector delivery, adenovirus vector delivery, retrovirus vector delivery or plasmid vector delivery in these animal model experiments (see above).
• One dose of a ribozyme vector that constitutively expresses the ribozyme or one or more doses of a stable anti-B7-1 , B7-2, B7-3 and/or CD40 ribozymes or a transiently expressing ribozyme vector to donor APC, followed by infusion into the recipient may reduce the incidence of graft rejection.
• graft tissues may be treated as described above prior to transplantation.
• Triethylamine (13.4 ml, 100 mmol) was added dropwise to a stirred ice- cooled mixture of 1 ,2,4-triazole (6.22g, 90 mmol) and phosphorous oxychloride (1.89 ml, 20 mmol) in 50 ml of anhydrous acetonitrile.
• phosphorous oxychloride (1.89 ml, 20 mmol) in 50 ml of anhydrous acetonitrile.
• 2',3',5'-tri-0-Benzoyl-6-methyl uridine 5.7g, 10 mmol
• acetonitrile 5.7g, 10 mmol
• the residue was dissolved in chloroform and washed with water, saturated aq sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and the solvent was removed in vacuo. The residue was dissolved in 100 ml of 1 ,4-dioxane and treated with 50 mL of 29% aq NH4OH overnight. The solvents were removed in vacuo. The residue was dissolved in the in the mixture of pyridine (60 mL) and methanol (10 mL), cooled to -15°C and ice-cooled 2M aq solution of sodium hydroxide was added under stirring.
• Triethylamine (23.7 ml, 170.4 mmol) was added dropwise to a stirred ice- cooled mixture of 1 ,2,4-triazole (10.6g, 153.36 mmol) and phosphorous oxychloride (3.22 ml, 34.08 mmol) in 100 ml of anhydrous acetonitrile.
• phosphorous oxychloride (3.22 ml, 34.08 mmol) in 100 ml of anhydrous acetonitrile.
• 2 ⁇ 3'-di-0-Acetyl-5'-0-Dimethoxytrityl-6- aza Uridine (7.13g, 11.36 mmol) in 40 ml of acetonitrile was added dropwise and the reaction mixture was stirred for 6 hours at room temperature. Then it was concentrated in vacuo to minimal volume (not to dryness).
• the residue was dissolved in chloroform and washed with water, saturated aq sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and the solvent was removed in vacuo.
• the residue was dissolved in 150 ml of 1 ,4-dioxane and treated with 50 mL of 29% aq NH4OH for 20 hours at room temperature. The solvents were removed in vacuo.
• the residue was purified by flash chromatigraphy on silica gel using linear gradient of MeOH (4% to 10%) in methylene chloride as an eluent to give 3.1g (50%) of azacytidine.
• Example 19 RNA cleavage activity of HHA ribozvme substituted with 6- methyl-Uridine Hammerhead ribozymes targeted to site A (see Fig. 31 ) were synthesized using solid-phase synthesis, as described above. U4 position was modified with 6-methyl-uridine.
• Substrate RNA is 5' end-labeled using [ ⁇ - 32 P] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme
• Trace amount ( ⁇ 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90°C for 2 min and snap-cooling on ice for 10 -15 min.
• the ribozyme and substrate are incubated, separately, at 37°C for 10 min in a buffer containing 50 mM Tris-HCl and 10 mM MgC.2-
• the reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 37°C. Aliquots of 5 ⁇ l are taken at regular intervals of time and the reaction is quenched by mixing with equal volume of 2X formamide stop mix. The samples are resolved on 20 % denaturing polyacrylamide gels.
• results are quantified and percentage of target RNA cleaved is plotted as a function of time.
• hammerhead ribozymes containing 6-methyl-uridine modification at U4 position cleave the target RNA efficiently.
• Example 20 RNA cleavage activity of HHB ribozvme substituted with 6- methvl-Uridine
• RNA cleavage reactions were carried out as described above. Referring to Fig. 34, hammerhead ribozymes containing 6-methyl-uridine modification at U4 and U7 positions cleave the target RNA efficiently.
• Example 21 RNA cleavage activity of HHC ribozvme substituted with 6- methyl-Uridine
• RNA cleavage reactions were carried out as described above. Referring to Fig. 36, hammerhead ribozymes containing 6-methyl-uridine modification at U4 positions cleave the target RNA efficiently.
• Example 22 Inhibition of Rat smooth muscle cell proliferation by 6-methyl-U substituted ribozvme HHA.
• HHA Hammerhead ribozyme
• site A a unique site within c- myb mRNA.
• c-myb protein has been shown to be essential for the proliferation of rat smooth muscle cell (Brown et al., 1992 J. Biol. Chem. 267, 4625).
• the ribozymes that cleaved site A within 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 described (Stinchcomb et al., supra).
• HHA ribozymes were complexed with lipids and delivered into rat smooth muscle cells. Serum-starved cells were stimulated as described by Stinchcomb et al., supra.
• RNA/lipid complex was added. The plates were incubated for 4 hours at 37°C. The medium was then removed and DMEM containing 10% FBS, additives and 10 ⁇ M bromodeoxyuridine (BrdU) was added. In some wells, FBS was omitted to determine the baseline of unstimulated proliferation. The plates were incubated at 37°C for 20-24 hours, fixed with 0.3% H 2 ⁇ 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.
• PrdU bromodeoxyuridine
• active ribozymes substituted with 6-methyl-U at position 4 of HHA were successful in inhibiting rat smooth muscle cell proliferation.
• a catalytically inactive ribozyme (inactive HHA), which has two base substitutions within the core (these mutations inactivate a hammerhead ribozyme; Stinchcomb et al., supra), does not significantly inhibit rat smooth muscle cell proliferation.
• Example 23 Inhibition of stromelysin production in human synovial fibroblast cells bv 6-methyl-U substituted ribozvme HHC.
• HHC Hammerhead ribozyme
• fibroblasts which produce stromelysin, are serum-starved overnight and ribozymes or controls are offered to the cells the next day. Cells were maintained in serum-free media. The ribozyme were applied to the cells as free ribozyme, or in association with various delivery vehicles such as cationic lipids (including TransfectamTM, LipofectinTM and LipofectamineTM), conventional liposomes, non-phospholipid liposomes or biodegradable polymers.
• cationic lipids including TransfectamTM, LipofectinTM and LipofectamineTM
• Interleukin- 1 ⁇ typically 20 units/ml
• stromelysin can then be monitored over a time course, usually up to 24 hours.
• HHC ribozyme containing 6-methyl-U modification caused a significant reduction in the level of stromelysin protein production. Catalytically inactive HHC had no significant effect on the protein level.
• ethyl acetate 3.9 g, 91% 4; Niedballa et al., Nucleic Acid Chemistry, Part 1 , Townsend, L.B. and Tipson, R.S., Ed.; J. Wiley & Sons, Inc.; New York, 1978, p 481-484); 10 (Niedballa and Vorbr ⁇ ggen, J. Org. Chem. 1974, 39, 3668-3671 ) was crystallized from ethanol (3.6 g, 84%).
• reaction mixtures were purified by the silica gel column chromatography (20- 50% gradient of ethyl acetate in hexanes) to enable faster moving 2'-0- TBDMSi isomers (68.5% and 55%, respectively) as colorless foams.
• Phosphoramidites 7 and 11 were incorporated into ribozymes and substrates using the method of synthesis, deprotection, purification and testing previously described (Wincott et al., 1995 supra). The average stepwise coupling yields were -98 %.
• compound 3 was prepared using the procedure analogous to that described by Czemecki and Ville, J. Org. Chem. 1989, 54, 610- 612. Contrary to their result, we succeeded in obtaining the title compound, by using the more acid resistant f-butyldiphenylsilyl group for 5-O-protection, instead of f-butyldimethylsilyl.
• Pseudouridine, 3-methyluridine or 2,4,6-trimethoxy benzene phosphoramidites were incorporated into ribozymes using solid phase synthesis as described by Wincott et al., 1995 supra.
• the ribozymes were deprotected using the standard protocol described above with the exception of ribozymes with pseudouridine.
• Pseudouridine-modified ribozymes were deprotected first by incubation at room temperature, instead of at 55°C, for 24 hours in a mixture of ethanolic ammonia (3:1).
• dihydrouridine phosphoramidite was synthesized based on the method described in Chaix et al., 1989 Nucleic Acid Res. 17, 7381- 7393 with certain improvements:
• Uridine (1 ; 10g, 41 mmoles) was dissolved in 200 ml distilled water and to the solution 2g of Rh (10% on alumina) was added. The slurry was brought to 60 psi of hydrogen, and hydrogenation was continued for 16hrs. Reaction was monitored by disappearance of UV absorbing material. All of starting material was converted to dihdrouridine (DHU) and tetrahydrouridine (2:1 based on NMR). Tetrahydrouridine was not removed at this step.
• DHU dihdrouridine
• tetrahydrouridine 2:1 based on NMR
• Dihydrouridine (2; 10g, 41 mmoles) was dissolved in 400ml dry pyridine; dimethylaminopyridine (0.244g,2mmoles), triethylamine (7.93ml, 56mmoles), and dimethoxytritylchloride (16.3g, 48mmoles) were added and stirred under argon overnight.
• the reaction was quenched with 50ml methanol, extracted with 400ml 5% sodium bicarbonate, and then 400ml brine. The organic phase was dried over sodium sulphate, filtered, and then dried to a foam.
• iii. 5'-DMT-DHU (3; 9.0g, 16.4mmoles) was dissolved in 150ml dry THF.
• Pyridine (4.9ml, ⁇ Ommoles) and silver nitrate (3.35g, 19.7mmoles) were added at room temperature and stirred under argon for 10min., then tert.- butyldimethylsilylchloride (tBDMS-CI; 3.0g, 19.7mmoles) was added and the slurry was stirred under argon overnight.
• tBDMS-CI tert.- butyldimethylsilylchloride
• the dihydrouridine was incorporated into ribozymes using solid phase synthesis as described by Wincott et al., 1995 supra, with improvements- nuceloside-oxalyl-polystyrene derivatized support (Alul et. al. Nucleic Acids Res.. 1991 , 19, 1527-1532) was used.
• the ribozyme containing the dihydrouridine substitution was deprotected using 30% methyl amine in anhydrous ethanol for 15 min. at room temperature and subsequent treatment with te/ -butyl-ammonium fluoride in anhydrous THF for 24 hrs. at room temperature.
• Example 28 Synthesis of 2-0-f-Butvldimethvlsilvl-5-0-dimethoxytrityl-3-0-(2- cvanoethvl- ⁇ /. ⁇ /-diiso p ropyiphosphoramidite)-1-deoxy-1-naphthyl- ⁇ -D- ribofuranose (7) phosphoramidites
• This phosphoramidite is incorporated into ribozymes using solid phase synthesis as described by Wincott et al., 1995 supra.
• the ribozyme containing naphthyl substitution was deprotected using the standard protocol described above.
• Example 29 Synthesis of 2-0-?-Butyldimethylsilyl-5-C-Dimethoxytrityl-3-Q-(2- Cvanoethyl- ⁇ /. ⁇ /-diisopropylphosphoramidite)-1-Deoxy-1-(p-Aminophenyl.- ⁇ - D-Ribofuranose phosphoramidites
• the title compound was prepared from 6 in an identical manner as for the synthesis of deblocked phenyl analog; (82% overall yield for 5'-0- desilylation and the cleavage of 2',3'-0-isopropylidene group).
• This phosphoramidite is incorporated into ribozymes using solid phase synthesis as described by Wincott et al., 1995 supra.
• the ribozyme containing aminophenyl substitution was deprotected using the standard protocol described above.
• RNA cleavage reactions were carried out as described above. Referring to
• HH-B ribozymes with either pyridin-4-one or phenyl substitution at position 7 were further characterized ( Figure 44). It appears that HH-B ribozyme with pyridin- 4-one modification at position 7 cleaves RNA with a 10 fold higher kcat when compared to a ribozyme with a U at position 7 (compare Figure 44 A with 44 B). HH-B ribozyme with a phenyl group at position 7 cleaves RNA with a 3 fold higher kcat when compared to a hammerhead ribozyme with U at position 7 (see Figure 44C).
• Table D2 is a summary of specified catalytic parameters (t and ts) on short substrates in vitro, and stabilities of the noted modified catalytic nucleic acids in human serum.
• U4 and U7 refer to the uracil bases noted in Figure 1. Modifications at the 2'-position are shown in the table.
• Figure 47 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided.
• the preferred sequence of a hammerhead ribozyme in a 5'- to 3'-direction of the catalytic core is CUGANGAGfbase paired withJCGAAA.
• the use of 2'-C-alkyl substituted nucleotides that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described.
• Ribozymes from Figure 47 and Table D2 were synthesized and assayed for catalytic activity and nuclease resistance. With the exception of entries 8 and 17, all of the modified ribozymes retained at least 1/10 of the wild-type catalytic activity. From Table D2, all 2'-modified ribozymes showed very large and significant increases in stability in human serum (shown) and in the other fluids described below (Example 3, data not shown). The order of most aggressive nuclease activity was fetal bovine serum > human serum > human plasma > human synovial fluid. As an overall measure of the effect of these 2'- substitutions on stability and activity, a ratio ⁇ was calculated (Table D2). This ⁇ value indicated that all modified ribozymes tested had significant, >100 -
• compound 37 may be used as a general intermediate to prepare derivatized 2'-C-alkyl phosphoramidites, where X is CH 3 , or an alkyl, or other group described above.
• X is CH 3 , or an alkyl, or other group described above.
• the following are other non-limiting examples showing the synthesis of nucleic acids using 2'-C-alkyl substituted phosphoramidites, the syntheses of the amidites, their testing for enzymatic activity and nuclease resistance. These examples are diagrammed in Figs 48-54.
• the method of synthesis used generally follows the procedure for normal RNA synthesis as described in Usman, N.; Ogilvie.K.K.; Jiang, M.-Y.; Cedergren, R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe, S.A.; Franklyn.C; Usman, N. Nucleic Acids Res. 1990, 18, 5433- 5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 10, 12, 17, 22, 31 , 18, 26, 32, 36 and 38).
• these 2'-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group I or Group II intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.
• RNA substrates 15-25-mers
• purified 5'-end labeled ribozymes ⁇ 36-m ⁇ rs
• Ribozyme stock solutions were 1 mM, 200 nM, 40 nM or 8 nM and the final substrate RNA concentrations were - 1 nM.
• Total reaction volumes were 50 mL.
• Example 35 3'.5'-Q-(Tetraisopropyl-disiloxane-1.3-diy ⁇ -2'-0-Phenoxythio- carbonyl-Uridine (7.
• Example 36 3'.5'-0-(Tetraisopropyl-disiloxane-1.3-divl.-2'-C-Allvl -Uridine (8.
• Example 38 5'-0-Dimethoxytrityl-2'-C-Allyl-Uridine 3'-(2-Cyanoethyl N.N- diisopropvlphosphoramidite.
• Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a stirred ice-cooled mixture of 1 ,2,4-triazole (5.66 g, 81.99 mmol) and phosphorous oxychloride (0.86 mL, 9.11 mmol) in 50 mL of anhydrous acetonitrile.
• a solution of 3',5'-0-(tetraisopropyl-disiloxane-1 ,3-diyl)- 2'-C-allyl uridine (2.32 g, 4.55 mmol) in 30 mL of acetonitrile was added dropwise and the reaction mixture was stirred for 4 h at room temperature.
• the reaction was concentrated in vacuo to a minimal volume (not to dryness). The residue was dissolved in chloroform and washed with water, saturated aq. sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and the solvent was removed in vacuo. The resulting foam was dissolved in 50 mL of 1 ,4-dioxane and treated with 29% aq. NH4OH overnight at room temperature. TLC (chloroform:methanol / 9:1) showed complete conversion of the starting material. The solution was evaporated, dried by coevaporation with anhydrous pyridine and acetylated with acetic anhydride (0.52 mL, 5.46 mmol) in pyridine overnight.
• Example 45 2'-Deoxy-2'-Difluoromethylene-3'.5'-Q-(Tetraisopropyldisilox- ane-1.3-div -Uridine
• Triethylamine (4.8 mL, 34 mmol) was added to a solution of POCI3 (0.65 mL, 6.8 mmol) and 1 ,2,4-triazole (2.1 g, 30.6 mmol) in acetonitrile (20 mL) at 0 °C.
• a solution of 2'-deoxy-2'-methylene-3',5'-0-(tetraisopropyldisiloxane-1 ,3- diyl) uridine 19 (1.65 g, 3.4 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at room temperature for 4 h.
• Example 50 1 -(2'-Deoxy-2'-Methylene-5'-0-Dimethoxytrityl- ⁇ -D-ribofurano- syl)-4- ⁇ /-Acetyl-Cvtosine 21
• Example 51 1 -(2'-Deoxy-2'-Methylene-5'-0-Dimethoxytrityl- ⁇ -D-ribof urano- syl)-4- ⁇ /-Acetyl-Cytosine 3'-(2-Cvanoethyl- ⁇ /. ⁇ /-diisopropylphosphoramidite) 122)
• Example 52 2'-Deoxy-2'-Difluoromethvlene-3'.5'-Q-(Tetraisopropvl disiloxane-1.3-divl)-4- ⁇ /-Acetvl-Cvtidine (24)
• Et ⁇ N (6.9 mL, 50 mmol) was added to a solution of POCI3 (0.94 mL, 10 mmol) and 1 ,2,4-triazole (3.1 g, 45 mmol) in acetonitrile (20 mL) at 0 °C.
• Example 54 1-(2'-Deoxy-2'-Difluoromethylene-5'-Q-Dimethoxytrityl- ⁇ -D- ribofuranosyl)-4- ⁇ /-Acetylcvtosine 3'-(2-cvanoethyl-N.N-diisopropylphosphor- amidite) (26)
• Acetic anhydride (4.6 mL) was added to a solution of 3',5'-0-(tetraiso- propyldisiloxane-1 ,3-diyl)-6- ⁇ /-(4-f-butylbenzoyl)-adenosine (Brown, J.; Christodolou, C; Jones.S.; Modak.A.; Reese, C; Sibanda.S.; Ubasawa A. J. Chem .Soc. Perkin Trans. / 1989, 1735) (6.2 g, 9.2 mmol) in DMSO (37 mL) and the resulting mixture was stirred at room temperature for 24 h. The mixture was concentrated in vacuo.
• Example 56 2 , -Deoxv-2 , -methvlene-3'.5'-Q-(Tetraisopropvldisiloxane-1.3- diyl)-6- ⁇ /-(4-f-Butvlbenzovl)-Adenosine (29)
• Example 60 2'-Deoxy-2'-Difluoromethylene-3'.5'-0-(Tetraisopropyldisilox- ane-1.3-diyl)-6- ⁇ /-(4-f-Butylbenzoyl .-Adenosine
• 6- ⁇ /-(4-f-butylbenzoyl)-adenosine (4.1 g, 6.4 mmol) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-difluoromethylene-6- ⁇ /-(4-f-butylbenzoyl)- adenosine (2.3 g, 4.9 mmol, 77%) was eluted with 20% MeOH in CH2CI2.
• Et3N 3 HF (3 mL) was added to a solution of 2'-deoxy-2'-methoxy- carboxylmethylidine-3',5'-0-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 33 (5 g, 9.3 mmol) dissolved in CH2CI 2 (20 mL) and Et3N (15 mL). The resulting mixture was evaporated in vacuo after 1 h and chromatographed on a silica gel column eluting 2'-deoxy-2'-methoxycarbonylmethylidine-uridine 34 (2.4 g, 8 mmol, 86%) with THF:CH 2 CI 2 / 4: 1.
• Example 68 2'-Deoxy-2'-Carboxymethylidine-3'.5'-0-(Tetraisopropyldi- siloxane-1.3-divl)-Uridine 37
• Example 69 Synthesis of 2'-C-allyl-U phosphoramidite from 5'-0-DMT-3'-Q- TBDMS-Uridine .
• Example 70 Synthesis of 5'-0-Dimethoxytrityl-2'-Q-Phenoxythiocarbonyl-3'- O-t-bytuldimethylsilyl-uridine 11.
• Example 72 Synthesis of 5'-0-Dimethoxytrityl-2'-C-Allyl Uridine (4) from 5'-Q- Dimethoxytrityl-2'-C-Allyl-3'-0-t-bvtuldimethyl-silyl-uridine (12).
• alkyl substituted nucleotides of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage or antisense situations.
• Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure. Administration of such oligonucleotides is by standard procedure. See Sullivan et al. PCT WO 94/02595.
• Example 73 Synthesis of Hammerhead Ribozymes Containing 2'-Q- alkylthioalkylnucleotides & Other Modified Nucleotides
• the method of synthesis follows the procedure for normal RNA synthesis as described in Usman, N.; Ogilvie.K.K.; Jiang,M.-Y.; Cedergren, RJ. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe.S.A.; Franklyn.C; Usman, N. Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
• 2'-0-alkylthioalkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group I or Group II intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefqre, of general use in any nucleic acid structure.
• Example 75 General procedure for the synthesis of 2'-Q-methylthiomethyl nucleosides (3) Referring to Figure 55, to a stirred ice-cooled solution of the mixture of base-protected 3 ⁇ 5'-0-(tetraisopropyldisiloxane-1 ,3-diyl) nucleoside (2) (7 mmol), methyl disulfide (70 mmol), 2,6-lutidine (7 mmol) in methylene chloride (100 mL) or mixture methylene chloride - acetonitrile (1 :1 ) under positive pressure of argon, solution of benzoyl peroxide (28 mmol) in methylene chloride was added dropwise during 1 hour.
• 5'-0-Dimethoxytrityl-2'0-Methylthiomethyl- Nucleosides (6) may also be synthesized using 5'-0-Dimethoxytrityl-3'-0- 1- Butyl-dimethy-lsilyl Nucleosides (4) as the starting material.
• Compound 4 is commercially available as a by-product during RNA phosphoramidite synthesis.
• Compond 4 is converted in to 3'-0-t-butyldimethylsilyl-2'-0- methylthiomethyl nucleoside 5, as described under example 3.
• Example 77 5'-0-Dimethoxytrityl-2'-Q-Methylthiomethyl-Nucleosides-3'-(2- Cvanoethyl-N.N-diisopropylphosphoroamidites) (7)
• Example 78 General procedure for the synthesis of 2'-Q-Methylthiophenyl nucleosides .
• Example 80 5'-0-Dimethoxvtrityl-2'-0-Methvlthiophenvl-Nucleosides-3'-(2- Cvanoethvl N.N-diisopropvlphosphoroamidites)
• Substrate RNA is 5' end-labeled using [ ⁇ - 32 P] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace amount ( ⁇ 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90°C for 2 min and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 37°C for 10 min in a buffer containing 50 mM Tris-HCl and 10 mM MgCI 2 .
• the reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 37°C. Aliquots of 5 ⁇ l are taken at regular intervals of time and the reaction is quenched by mixing with equal volume of 2X formamide stop mix. The samples are resolved on 20 % denaturing polyacrylamide gels. The results are quantified and percentage of target RNA cleaved is plotted as a function of time. Referring to Figure 57, hammerhead ribozymes containing 2'-0- methylthiomethyl modifications at various positions cleave the target RNA efficiently. Surprisingly, all the 2'-0-methylthiomethyl -substituted ribozymes cleaved the target RNA more efficiently compared to the control hammerhead ribozyme.
• Such non-nucleotides can be located in the binding arms, core or the loop adjacent stem II of a hammerhead type ribozyme. Those in the art following the teachings herein can determine optimal locations in these regions. Surprisingly, abasic moieties can be located in the core of such a ribozyme.
• Example 83 RNA cleavage assay in vitro
• Ribozymes and substrate RNAs were synthesized as described above.
• Substrate RNA was 5' end-labeled using [ ⁇ -8 2 P] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace amount ( ⁇ 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme were denatured and renatured separately by heating to 90°C for 2 min and snap-cooling on ice for 10 -15 min.
• the ribozyme and substrate were incubated, separately, at 37°C for 10 min in a buffer containing 50 mM Tris-HCl and 10 mM MgCI 2 .
• the reaction was initiated by mixing the ribozyme and substrate solutions and incubating at 37°C. Aliquots of 5 ⁇ l are taken at regular intervals of time and the reaction quenched by mixing with an equal volume of 2X formamide stop mix. The samples were resolved on 20 % denaturing polyacrylamide gels. The results were quantified and percentage of target RNA cleaved is plotted as a function of time.
• FIG. 59 there is shown the general structure of a hammerhead ribozyme targeted against site B (HH-B) with various bases numbered.
• Various substitutions were made at several of the nucleotide positions in HH-B.
• substitutions were made at the U4 and U7 positions marked as X4 and X7 and also in loop II in the positions marked by an X.
• the RNA cleavage activity of these substituted ribozymes is shown in the following figures.
• Figure 61 shows cleavage by an abasic substituted U4 and an abasic substituted U7.
• abasic substitution at U4 or U7 does not significantly affect cleavage activity.
• inclusion of all abasic moieties in stem II loop does not significantly reduce enzymatic activity as shown in Figure 62. Further, inclusion of a 3' inverted deoxyribose does not inactivate the RNA cleavage activity as shown in Figure 63.
• HH-A Hammerhead ribozyme
• site A a unique site within c-myb mRNA.
• c-myb protein has been shown to be essential for the proliferation of rat smooth muscle cell (Brown et al., 1992 J. Biol. Chem. 267, 4625).
• Rat vascular smooth muscle cells were isolated and cultured as described (Stnchcomb et al., supra). These primary rat aortic smooth muscle cells (RASMC) were plated in a 24-well plate (5x10 3 cells/well) and incubated at 37°C in the presence of Dulbecco's Minimal Essential Media (DMEM) and 10% serum for -16 hours.
• DMEM Dulbecco's Minimal Essential Media
• LFA lipofectamine
• Ribozyme ⁇ FA complex was incubated with serum-starved RASMC cells for four hours at 37°C. Following the removal of ribozyme ⁇ FA complex from cells (after 4 hours), 10% serum was added to stimulate smooth cell proliferation. Bromo-deoxyuridine (BrdU) was added to stain the cells. The cells were stimulated with serum for 24 hours at 37°C. Following serum-stimulation, RASMC cells were quenched with hydrogen peroxide (0.3% H2O2 in methanol) for 30 min at 4°C. The cells were then denatured with 0.5 ml 2N HCI for 20 min at room temperature.
• PrdU Bromo-deoxyuridine
• Horse serum (0.5 ml) was used to block the cells at 4°C for 30 min up to -16 hours.
• the RASMC cells were stained first by treating the cells with anti-BrdU (primary) antibody at room temperature for 60 min. The cells were washed with phosphate-buffered saline (PBS) and stained with biotinylated affinity- purified anti-mouse IgM (Pierce, USA) secondary antibody. The cells were counterstained using avidin-biotinylated enzyme complex (ABC) kit (Pierce, USA).
• PBS phosphate-buffered saline
• biotinylated affinity- purified anti-mouse IgM Pieris, USA
• the ratio of proliferating:non-proliferating cells was determined by counting stained cells under a microscope. Proliferating RASMCs will incorporate BrdU and will stain brown. Non-proliferating cells do not incorporate BrdU and will stain purple.
• FIG 64 there is shown a ribozyme which cleaves the site A referred to as HH-A. Substitutions of abasic moieties in place of U4 as shown in Figure 65 provided active ribozyme as shown in Figure 66 using the above- noted rat aortic smooth muscle cell proliferation assay.
• the method of this invention generally features HPLC purification of ribozymes.
• An example of such purification is provided below in which a synthetic ribozyme produced on a solid phase is blocked. This material is then released from the solid phase by a treatment with methanolic ammonia, subsequently treated with tetrabutylammonium fluoride, and purified on reverse phase HPLC to remove partially blocked ribozyme from "failure" sequences.
• "failure" sequences are RNA molecules which have a nucleotide base sequence shorter to that of the desired enzymatic RNA molecule by one or more of the desired bases in a random manner, and possess free terminal 5'-hydroxyl group.
• This terminal 5'-hydroxyl in a ribozyme with the correct sequence is still blocked by lipophilic dimethoxytrityl group.
• enzymatic RNA is purified, it is deblocked by a standard procedure, and passed over the same or a similar HPLC reverse phase column to remove other contaminating components, such as other RNA molecules or nucleotides or other molecules produced in the deblocking and synthetic procedures.
• the resulting molecule is the native enzymatically active ribozyme in a highly purified form.
• Solid phase synthesis was carried out on either an ABI 394 or 380B DNA/RNA synthesizer using the standard protocol provided with each machine. The only exception was that the coupling step was increased from 10 to 12 minutes. The phosphoramidite concentration was 0.1 M. Synthesis was done on a 1 ⁇ mol scale using a 1 ⁇ mol RNA reaction column (Glen Research). The average coupling efficiencies were between 97% and 98% for the 394 model and between 97% and 99% for the 380B model, as determined by a calorimetric measurement of the released trityl cation. The final 5'-DMT group was not removed.
• the ribozymes were cleaved from the CPG support, and the base and phosphotriester moieties were deprotected in a sterile vial by incubation in dry ethanolic ammonia (2 mL) at 55 °C for 16 hours. The reaction mixture was cooled on dry ice. Later, the cold liquid was transferred into a sterile screw cap vial and lyophilized.
• the ribozymes were purified in two steps by HPLC on a C4 300 A 5 ⁇ m DeltaPak column in an acetonitrile gradient.
• the first step was a separation of 5'-DMT-protected ribozyme(s) from failure sequences lacking a 5'-DMT group.
• Solvents used for this step were: A (0.1 M triethylammonium acetate, pH 6.8) and B (acetonitrile).
• the elution profile was: 20% B for 10 minutes, followed by a linear gradient of 20% B to 50% B over 50 minutes, 50% B for 10 minutes, a linear gradient of 50% B to 100% B over 10 minutes, and a linear gradient of 100% B to 0% B over 10 minutes.
• the second step was a purification of a completely deprotected, i.e. following the removal of the 5'-DMT group, ribozyme by a treatment with 2% trifluoroacetic acid or 80% acetic acid on a C4 300 A 5 ⁇ m DeltaPak column in an acetonitrile gradient.
• Solvents used for this second step were: A (0.1 M Triethylammonium acetate, pH 6.8) and B (80% acetonitrile, 0.1 M triethylammonium acetate, pH 6.8).
• the elution profile was: 5% B for 5 minutes, a linear gradient of 5% B to 15% B over 60 minutes, 15% B for 10 minutes, and a linear gradient of 15% B to 0% B over 10 minutes.
• the fraction containing ribozyme which is in the triethylammonium salt form, was cooled and lyophilized on a SpeedVac. Solid residue was dissolved in a minimal amount of ethanol and ribozyme in sodium salt form was precipitated by addition of sodium perchlorate in acetone. (K + or Mg 2+ salts can be produced in an equivalent manner.) The ribozyme was collected by centrifugation, washed three times with acetone, and lyophilized.
• the polymer-bound oligonucleotide either trityl-on or off, was suspended in a solution of ethylamine (EA) @ 25-55 °C for 10-30 min to remove the exocyclic amino protecting groups (see Figure 67).
• EA ethylamine
• the supernatant was removed from the polymer support.
• the support was washed with 1.0 mL of EtOH:MeCN:H 2 0/3:1 :1 , vortexed and the supernatant was then added to the first supernatant.
• the combined supernatants, containing the oiigoribonucleotide were dried to a white powder.
• Table EVII is a summary of the results obtained using the improvements outlined in this application for base deprotection. From this data it is evident
• the second step of the deprotection of RNA molecules may be accomplished by removal of the 2'-hydroxyl alkylsilyl protecting group using TBAF for 8-24 h (Usman et al. J. Am. Chem. Soc. 1987, 709, 7845-7854). Applicant has determined that the use of anhydrous TEA»HF in N- methylpyrrolidine (NMP) for 0.5-1.5 h @ 55-65 °C gives equivalent or better results.
• NMP N- methylpyrrolidine
• Ribozymes were synthesized in two parts and tested without ligation for catalytic activity. Referring to Fig. 72, the cleavage activity of the half ribozymes containing between 5 and 8 base pairs stem Us at 40 nM under single turnover conditions was comparable to that of the full length oligomer as shown in Figs. 73 and 74. The same half ribozymes were synthesized with suitable modifications at the nascent stem II loop to allow for crosslinking. The halves were purified and chemically ligated, using a variety of crosslinking methods. The resulting full length ribozymes (see Fig. 71 ) exhibited similar cleavage activity as the linearly synthesized full length oligomer as shown in Fig. 74.
• ribozyme had a ribose group. This was oxidatively cleaved with Nal ⁇ 4 and reacted with the 3' half of the ribozyme having an amino group under reducing conditions.
• the resulting ribozyme consisted of the two half ribozyme linked by a morpholino group.
• a 5' half of ribozyme was provided with a carboxyl group at its 2' position and was coupled with an amine containing 3' half ribozyme.
• the provision of a coupling reagent resulted in a full-length ribozyme having an amide bond.
• RNA was transcribed, using T7 RNA polymerase, in a standard transcription buffer in the presence of [ ⁇ - 32 P]CTP.
• the reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol (25:1), precipitated with isopropanol and washed with 70% ethanol.
• the dried pellet was resuspended in 20 ⁇ l DEPC- treated water and stored at -20°C.
• Unlabeled ribozyme (200 nM) and internally labeled 1.8 KB substrate RNA ( ⁇ 10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris-HCl pH 7.5 and 10 mM MgCI 2 ) by heating to 90°C for 2 min. and slow cooling to 37°C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37°C. Aliquots of 5 ⁇ l were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).
• Gene therapy represents a potential alternative strategy, where antiviral genes are stably transferred into susceptible cells.
• Such gene therapy approaches have been termed "intracellular immunization" since cells expressing antiviral genes become immune to viral infection (Baltimore, 1988 Nature 335, 395-396).
• Numerous forms of antiviral genes have been developed, including protein-based antivirals such as transdominant inhibitory proteins (Malim et al., 1993 J. Exp. Med., Bevec et al., 1992 P.N.A.S. (USA) 89, 9870-9874; Bruer et al., 1993 J. Virol.
• RNA based molecules such as antisense RNAs, decoy RNAs, agonist RNAs, antagonist RNAs, therapeutic editing RNAs and ribozymes. RNA is not immunogenic; therefore, cells expressing such therapeutic RNAs are not susceptible to immune eradication.
• a transcription unit termed U6-S35, is designed that contains the characteristic intramolecular stem of a S35 motif (see Figure 76). As shown in Figure 77, 78 and 79 a desired RNA (e.g. ribozyme) can be inserted into the indicated region of U6-S35 chimera. This construct is under the control of a type 3 pol III promoter, such as a mammalian U6 small nuclear RNA (snRNA) promoter (see Fig. 75). U6-S35-HHI and U6-S35-HHII are non-limiting examples of the U6-S35 chimera.
• snRNA mammalian U6 small nuclear RNA
• applicant has constructed a stable, active ribozyme RNA driven from a eukaryotic U6 promoter (Fig. 78).
• a stable, active ribozyme RNA driven from a eukaryotic U6 promoter For stability, applicant incorporated a S35 motif as described in Fig. 76 and Fig. 77.
• a ribozyme sequence is inserted at the top of the stem, such that the ribozyme is separated from the S35 motif by an unstructured spacer sequence (Fig. 77, 78, 79).
• the spacer sequence can be customized for each desired RNA sequence.
• U6-S35 chimera is meant to be a non-limiting example and those skilled in the art will recognize that the structure disclosed in the figures 77, 78 and 79 can be driven by any of the known RNA polymerase promoters and are within the scope of this invention. All that is necessary is for the 5' region of a transcript to interact with its 3' region to form a stable intramolecular structure (S35 motif) and that the S35 motif is separated from the desired RNA by a stretch of unstructured spacer sequence. The spacer sequence appears to improve the effectiveness of the desired RNA.
• unstructured is meant lack of a secondary and tertiary structure such as lack of any stable base-paired structure within the sequence itself, and preferably with other sequences in the attached RNA.
• spacer sequence is meant any unstructured RNA sequence that separates the S35 domain from the desired RNA. The spacer sequence can be greater than or equal to one nucleotide.
• U6-S35-HHI ribozyme RNA was synthesized using T7 RNA polymerase.
• HHI RNA was chemically synthesized using RNA phosphoramidite chemistry as described in Wincott et al., 1995 Nucleic Acids Res.
• the ribozyme RNAs were gel-purified and the purified ribozyme RNAs were heated to 55°C for 5 min.
• Target RNA used was -650 nucleotide long.
• lnternally- 3 P-labeled target RNA was prepared as described above.
• the target RNA was pre ⁇ heated to 37°C in 50 mM Tris.HCI, 10 mM MgCt ⁇ and then mixed at time zero with the ribozyme RNAs (to give 200 nM final concentration of ribozyme). At appropriate times an aliquot was removed and the reaction was stopped by dilution in 95% formamide. Samples were resolved on a denaturing urea- polyacrylamide gel and products were quantitated on a phospholmager®.
• the U6-S35-HHI ribozyme chimera cleaved its target RNA as efficiently as a chemically synthesized HHI ribozyme.
• the U6-S35-HHI ribozyme chimera may be more efficient than the synthetic ribozyme.
• Actinomycin D assay was used to measure accumulation of the transcript in mammalian cells.
• Cells were transfected overnight with plasmids encoding the appropriate transcription units (2 ⁇ g DNA well of 6 well plate) using calcium phosphate precipitation method (Maniatis et al., 1982 Molecular Cloning Cold Spring Harbor Laboratory Press, NY). After the overnight transfection, media was replaced and the cells were incubated an additional 24 hours. Cells were then incubated in media containing 5 ⁇ g/ml Actinomycin D.
• the U6-S35-HHII ribozyme shown in Figure 79 is fairly stable in 293 mammalian cells with an approximate half-life of about 2 hours.
• a transcription unit consisting of a wild type VA1 sequence with two modifications: a "S35-like” motif extends from a loop in the central domain ( Figure 82); the 3' terminus is changed such that there is a more complete interaction between the 5' and the 3' region of the transcript (specifically, an "A-C” bulge is changed to an "A-U base pair and the termination sequence is part of the stem of S35 motif).
• VA1-S35-ribozvme transcripts An Actinomycin D assay was used to measure accumulation of the transcript in mammalian cells as described above.
• the VA1 -S35-chimera shown in Figure 83A, has approximately 10-fold higher stability in 293 mammalian cells compared to VA1 -chimera, shown in Figure 25B that lacks the intramolecular S35 motif.
• RNAs, decoys can be readily inserted into the indicated U6-S35 or VA1 -S35 chimera to achieve therapeutic levels of RNA expression in mammalian cells.
• 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 stromolysin, B7-1 , B7-2, B7-3 and/or CD40 or other RNAs 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 B7-1 , B7-2, B7-3 and/or CD40 or other RNA related conditions. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
• ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay.
• the first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample.
• synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme 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.
• 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., B7-1 , B7-2, B7-3 and/or CD40 is adequate to establish risk.
• 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.
• RNAseP RNA M1 RNA
• Size -290 to 400 nucleotides.
• RNA portion of a ribonucleoprotein enzyme Cleaves tRNA precursors to form mature tRNA.
• RNA RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus which uses RNA as the infectious agent ( Figure 3).
• HDV Hepatitis Delta Virus
• NO. 187 996 ACUUUUQGOGCAAADC CXXXaOGAAGCUUGA 3D.
• NO. 188 1000 UUGG ⁇ CAAAUCCCUC AGGAAGCDUGAACCU 3D.
• NO. 189 1009 AIXXrtXfiGGAAGCUU G ⁇ KX ⁇ I3M-JLX3CI- 3D.
• NO. 191 191

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