Title:  Ribozyme therapy for the treatment of proliferative skin and eye diseases

United States Patent:  6,770,633

Issued:  August 3, 2004

Inventors:  Robbins; Joan M. (San Diego, CA); Tritz; Richard (San Diego, CA)

Assignee:  Immusol, Inc. (San Diego, CA)

Appl. No.:  696791

Filed:  October 25, 2000

Abstract

As an effective therapy for proliferative skin and eye diseases such as psoriasis and proliferative diabetic retinopathy, this invention provides ribozymes and ribozyme delivery systems which cleave RNA encoding cytokines involved in inflammation, matrix metalloproteinases, a cyclin, a cell-cycle dependent kinase, a growth factor, or a reductase.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides ribozymes and ribozyme delivery systems which are able to inhibit the mechanisms involved in proliferation of cells associated with proliferative skin and eye diseases such as psoriasis, PDR and scarring. Accordingly, the present invention ribozymes are provided which are suitable for treating scarring and proliferative diseases by inhibiting a cytokine involved in inflammation, a matrix metalloproteinase involved in extracellular matrix elaboration, a cyclin, a cell-cycle dependent kinase, a growth factor involved in cell cycle regulation, or a reductase.

Within one aspect of the invention, such methods generally comprise the step of administering to a patient a therapeutically effective amount of ribozyme which cleaves RNA encoding a cytokine involved in inflammation, a matrix metalloproteinase, a cyclin, a cell-cycle dependent kinase, a growth factor, or a reductase such that said proliferative skin disease is treated. Within a related aspect, methods of treating proliferative skin disease are provided comprising the step of administering to a patient an effective amount of nucleic acid molecule comprising a promoter operably linked to a nucleic acid segment encoding a ribozyme which cleaves RNA encoding a cytokine involved in inflammation, a matrix metalloproteinase, a cyclin, a cell-cycle dependent kinase, a growth factor, or a reductase such that said proliferative skin disease is treated. Representative examples of proliferative skin diseases include psoriasis, atopic dermatitis, actinic keratosis, squamous or basal cell carcinoma, viral or seborrheic wart.

Within other aspects, such methods generally comprise the step of administering to a patient a therapeutically effective amount of ribozyme which cleaves RNA encoding a cytokine involved in inflammation, a matrix metalloproteinase, a cyclin, a cell-cycle dependent kinase, or a growth factor such that said proliferative eye disease is treated. Within a related aspect, methods of treating proliferative eye disease are provided comprising the step of administering to a patient an effective amount of nucleic acid molecules comprising a promoter operably linked to a nucleic acid segment encoding a ribozyme which cleaves RNA encoding a cytokine involved in inflammation, a matrix metalloproteinase, a cyclin, a cell-cycle dependent kinase, a growth factor, or a reductase such that said proliferative eye disease is treated. Representative examples of proliferative eye diseases include proliferative diabetic retinopathy, proliferative vitreoretinopathy, proliferative sickle cell retinopathy, retinopathy of prematurity and retinal detachment.

Within other aspects, methods are provided for treating or preventing scarring, comprising administering to a patient a therapeutically effective amount of ribozyme which cleaves RNA encoding a cytokine involved in inflammation, a matrix metalloproteinase, a cyclin, a cell-cycle dependent kinase, a growth factor, or a reductase such that said scarring is treated or prevented. Within another related aspect, methods are provided for treating or preventing scarring, comprising administering to a patient an effective amount of nucleic acid molecule comprising a promoter operably linked to a nucleic acid segment encoding a ribozyme which cleaves RNA encoding a cytokine involved in inflammation, a matrix metalloproteinase, a cyclin, a cell-cycle dependent kinase, a growth factor, or a reductase such that said scarring is treated or prevented. Representative examples of diseases or injuries which cause scarring include keloids, adhesions (e.g., surgical adhesions), hypertrophic burn scars, and trauma (e.g., blunt trauma or surgical trauma).

Cyclins and cell-cycle dependent kinases are directly involved in the check point control of cell division leading to proliferation. Particularly preferred cyclins or cell-cycle dependent kinases include CDK1, CDK2, CDK4, Cyclin B1, Cyclin D and PCNA.

Infection or injury induces a complex cascade of events including activation of the clotting pathway, adherence of immune cells to the endothelium, induction of proliferation and migration of cells into the injury site to rapidly repair the insult. These responses are mediated by certain cytokines and growth factors released from the injured tissue and by hematopoetic cells. Chronic inflammation develops when cytokines and growth factors continue to be produced, resulting in an exaggerated healing response. Particularly preferred cytokines include the inflammatory cytokines interleukin 1 alpha and beta, interleukin 2, interleukin 6, interleukin 8, interferon gamma, and tumor necrosis factor. Particularly preferred growth factors include vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF).

Matrix metalloproteinases, along with their corresponding inhibitors, tissue inhibitors of metalloproteinases or TIMP, are involved in the repair of injured tissue. The balance between the proteinases and the inhibitors regulates the pattern and extent of wound healing. Excessive production of MMP or insufficient production of TIMP results in abnormal wound healing. Particularly preferred matrix metalloproteinases include MMP 1, MMP 2, MMP 3 and MMP 9.

Preferably, the ribozyme is a hammerhead or hairpin ribozyme, representative examples of which recognize the target site sequences set forth below and in the Examples. In preferred embodiments, the present invention also provides nucleic acid molecule encoding such ribozymes; further preferably, the nucleic acid is DNA or cDNA. Even further preferably, the nucleic acid molecule is under the control of a promoter to transcribe the nucleic acid.

In one embodiment of the invention, nucleic acid molecules are described which encode the ribozymes provided herein. Preferably, the vector is a plasmid, a virus, retrotransposon, a cosmid or a retrovirus. In one embodiment where the vector is a retroviral vector, the nucleic acid molecule encoding the ribozyme under the control of a promoter, which is preferably a pol III promoter, further preferably a human tRNA^Val promoter or an adenovirus VAl promoter, is inserted between the 5' and 3' long terminal repeat sequences of the retrovirus.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Prior to setting forth the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used hereinafter.

"Ribozyme" refers to a nucleic acid molecule which is capable of cleaving a specific nucleic acid sequence. Ribozymes may be composed of RNA, DNA, nucleic acid analogues (e.g., phosphorothioates), or any combination of these (e.g., DNA/RNA chimerics). Within particularly preferred embodiments, a ribozyme should be understood to refer to RNA molecules that contain anti-sense sequences for specific recognition, and an RNA-cleaving enzymatic activity.

"Ribozyme gene" refers to a nucleic acid molecule (e.g., DNA) consisting of the ribozyme sequence which, when transcribed into RNA, will yield the ribozyme.

"Vector" refers to an assembly which is capable of expressing a ribozyme of interest. The vector may be composed of either deoxyribonucleic acids ("DNA") or ribonucleic acids ("RNA"). Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase, hygromycin phosphotransferase or puromycin-N-acetyl-transferase. Additionally, depending on the host cell chosen and the vector employed, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences conferring inducibility of transcription, and selectable markers, may also be incorporated into the vectors described herein.

"Nucleic acid" or "nucleic acid molecule" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acids can be composed of monomers that are naturally-occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), or analogs of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid" also includes so-called "peptide nucleic acids," which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

"Isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a gene that has been separated from the genomic DNA of a eukaryotic cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.

"Promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.

As noted above, proliferative diseases such as psoriasis, PDR, and scarring affect many individuals and represent a major cost to the health care system. As discussed in more detail below, by interfering with cell-cycle control of cells which might otherwise proliferate, proliferative diseases such as psoriasis or PDR can be effectively treated. This invention accomplishes such by providing ribozymes and methods of using ribozymes that inhibit the activity of cytokines which are involved in inflammation, matrix metalloproteinases, cyclins, cell-cycle dependent kinases, growth factors, reductases, all of which directly or indirectly contribute to proliferation.

Cyclins and cell-cycle dependent kinases are directly involved in the signaling and synthesis that results in cells progressing through the cell cycle to replicate. Representative examples of suitable ribozyme targets include cdk1 ribozyme binding sites (SEQ ID NOS: 1-149); cdk2 ribozyme binding sites (SEQ ID NOS: 150-3010); cdk3 ribozyme binding sites (SEQ ID NOS: 302-405); cdk4 ribozyme binding sites (SEQ ID NOS: 406-526); cdk6 ribozyme binding sites (SEQ ID NOS: 527-665); cdk7 ribozyme binding sites (SEQ ID NOS: 666-866); cdk8 ribozyme binding sites (SEQ ID NOS: 867-1112); cdk-we-hu ribozyme binding sites (SEQ ID NOS: 1113-1408); cyclin A2 ribozyme binding sites (SEQ ID NOS: 1409-1614); cyclin C ribozyme binding sites (SEQ ID NOS: 1615-1819); cyclin D1 ribozyme binding sites (SEQ ID NOS: 1820-1889); cyclin D2 ribozyme binding sites (SEQ ID NOS: 1890-1975); cyclin D3 ribozyme binding sites (SEQ ID NOS: 1976-2053); cyclin E ribozyme binding sites (SEQ ID NOS: 2054-2318); cyclin F ribozyme binding sites (SEQ ID NOS: 2319-2561); cyclin G1 ribozyme binding sites (SEQ ID NOS: 2562-2787); cyclin H ribozyme binding sites (SEQ ID NOS: 2788-2964); cyclin A1 ribozyme binding sites (SEQ ID NOS: 2965-3257); cyclin B1 ribozyme binding sites (SEQ ID NOS: 3258-3478) ; cdc25 hs ribozyme binding sites (SEQ ID NOS: 3479-3854); PCNA HH ribozyme binding sites (SEQ ID NOS: 3855-4115); and chimeric hairpin ribozymes: SEQ ID NOS: 4116-4119).

As noted above, infection or injury induces a complex cascade of events including activation of the clotting pathway, adherence of immune cells to the endothelium, induction of proliferation and migration of cells into the injury site to rapidly repair the insult. These responses are mediated by certain cytokines and growth factors released from the injured tissue and by hematopoetic cells. Chronic inflammation develops when cytokines and growth factors continue to be produced, resulting in an exaggerated healing response. Particularly preferred cytokines include the inflammatory cytokines interleukin 1 alpha and beta, interleukin 2, interleukin 6, interleukin 8, interferon gamma, and tumor necrosis factor. Particularly preferred growth factors include vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF).

Matrix metalloproteinases, along with their corresponding inhibitors, tissue inhibitors of metalloproteinases or TIMP, are involved in a the repair of injured tissue. The balance between the proteinases and the inhibitors regulates the pattern and extent of wound healing. Excessive production of MMP or insufficient production of TIMP results in abnormal wound healing. Particularly preferred-matrix metalloproteinases include MMP 1, MMP 2, MMP 3 and MMP 9.

Ribozymes

As noted above, the present invention provides ribozymes having the ability to cleave or otherwise inhibit nucleic acid molecules which are either directly, or indirectly (e.g., they encode proteins) involved in cell-cycle control. Several different types of ribozymes may be constructed for use within the present invention, including for example, hammerhead ribozymes (Rossi, J. J. et al., Pharmac. Ther. 50:245-254, 1991) (Forster and Symons, Cell 48:211-220, 1987; Haseloff and Gerlach, Nature 328:596-600, 1988; Walbot and Bruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature 334:585, 1988; Haseloff et al., U.S. Pat. No. 5,254,678), hairpin ribozymes (Hampel et al., Nucl. Acids Res. 18:299-304, 1990, and U.S. Pat. No. 5,254,678), hepatitis delta virus ribozymes (Perrotta and Been, Biochem. 31:16, 1992), Group I intron ribozymes (Cech et al., U.S. Pat. No. 4,987,071) and RNase P ribozymes (Takada et al., Cell 35:849, 1983); (see also, WO 95/29241, entitled "Ribozymes with Product Ejection by Strand Displacement"; and WO 95/31551, entitled "Novel Enzymatic RNA Molecules."

Cech et al. (U.S. Pat. No. 4,987,071, issued Jan. 22, 1991) has disclosed the preparation and use of ribozymes which are based on the properties of the Tetrahymena ribosomal RNA self-splicing reaction. These ribozymes require an eight base pair target site and free guanosine (or guanosine derivatives). A temperature optimum of 50^oC. is reported for the endoribonuclease activity. The fragments that arise from cleavage contain 5'-phosphate and 3'-hydroxyl groups and a free guanosine nucleotide added to the 5'-end of the cleaved RNA.

In contrast to the ribozymes of Cech et al., particularly preferred ribozymes of the present invention hybridize efficiently to target sequences at physiological temperatures, making them suitable for use in vivo, and not merely as research tools (see column 15, lines 18 to 42, of Cech et al., U.S. Pat. No. 4,987,071). Thus, particularly preferred ribozymes for use within the present invention include hairpin ribozymes (for example, as described by Hampel et al., European Patent Publication No. 0 360 257, published Mar. 26, 1990) and hammerhead ribozymes. Briefly, the sequence requirement for the hairpin ribozyme is any RNA sequence consisting of NNNBN*GUC (N).lambda. (Sequence ID Nos. 4120-4124) (where x is any number from 6 to 10, N*G is the cleavage site, B is any of G, C, or U, and N is any of G, U, C, or A). Representative examples of recognition or target sequences for hairpin ribozymes are set forth below in the Examples. Additionally, the backbone or common region of the hairpin ribozyme can be designed using the nucleotide sequence of the native hairpin ribozyme (Hampel et al., Nucl. Acids Res. 18:299-304, 1990) or it can be modified to include a "tetraloop" structure that increases stability and catalytic activity (see Example 2; see also Yu et al., Virology 206:381-386, 1995; Cheong et al., Nature 346:680-682, 1990; Anderson et al., Nucl. Acids Res. 22:1096-1100, 1994).

The sequence requirement at the cleavage site for the hammerhead ribozyme is any RNA sequence consisting of NUH (where N is any of G, U, C, or A and H represents C, U, or A) can be targeted. Accordingly, the same target within the hairpin leader sequence, GUC, is useful for the hammerhead ribozyme. The additional nucleotides of the hammerhead ribozyme or hairpin ribozyme is determined by the target flanking nucleotides and the hammerhead consensus sequence (see Ruffner et al., Biochemistry 29:10695-10702, 1990). This information, along with the sequences and disclosure provided herein, enables the production of hairpin ribozymes of this invention.

The ribozymes of this invention, as well as DNA encoding such ribozymes and other suitable nucleic acid molecules, described in more detail below, can be chemically synthesized using methods well known in the art for the synthesis of nucleic acid molecules (see e.g., Heidenreich et al., J FASEB 70(1):90-6, 1993; Sproat, Curr. Opin. Biotechnol. 4(1):20-28, 1993). Alternatively, commercial suppliers such as Promega, Madison, Wis., USA, provide a series of protocols suitable for the production of nucleic acid molecules such as ribozymes.

Within one aspect of the present invention, ribozymes are prepared from a DNA molecule or other nucleic acid molecule (which, upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase. Accordingly, also provided by this invention are nucleic acid molecules, e.g., DNA or cDNA, coding for the ribozymes of this invention. When the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, the ribozyme can be produced in vitro upon incubation with the RNA polymerase and appropriate nucleotides. In a separate embodiment, the DNA may be inserted into an expression cassette, such as described in Cotten and Birnstiel, EMBO J. 8(12):3861-3866, 1989, and in Hempel et al., Biochemistry 28:4929-4933, 1989. A more detailed discussion of molecular biology methodology is disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.

During synthesis, the ribozyme can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase (Rossi et al., Pharmac. Ther. 50:245-254, 1991). In another embodiment, the ribozyme can be modified to a phosphothio-analog for use in liposome delivery systems. This modification also renders the ribozyme resistant to endonuclease activity. In yet another embodiment, the ribozyme can be modified to contain propanediol linkages or to incorporate 2'-O-methylated nucleotides.

Vectors

Use of ribozymes to treat proliferative skin diseases such as psoriasis or eczema involves introduction of functional ribozyme to the infected cell of interest. This can be accomplished by either synthesizing functional ribozyme in vitro prior to delivery, or, by delivery of DNA capable of driving ribozyme synthesis in vivo.

More specifically, within other aspects of the invention the ribozyme gene may be constructed within a vector which is suitable for introduction to a host cell (e.g., prokaryotic or eukaryotic cells in culture or in the cells of an organism). Appropriate prokaryotic and eukaryotic cells can be transfected with an appropriate transfer vector containing the nucleic acid molecule encoding a ribozyme of this invention.

To produce the ribozymes with a vector in vivo, nucleotide sequences coding for ribozymes are preferably placed under the control of a eukaryotic promoter such as pol III (e. g., tRNA or VA-1 from adenovirus), CMV, SV40 late, or SV40 early promoters. Within certain embodiments, the promoter may be a tissue or cell-specific promoter. Ribozymes may thus be produced directly from the transfer vector in vivo.

A wide variety of vectors may be utilized within the context of the present invention, including for example, plasmids, viruses, retrotransposons and cosmids. Representative examples include adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Yei et al., Gene Therapy 1:192-200, 1994; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J. Neurosci. 5(10):1287-1291, 1993), adeno-associated type 1 ("AAV-1") or adeno-associated type 2 ("AAV-2") vectors (see WO 95/13365; Flotte et al., PNAS 90(22):10613-10617, 1993), hepatitis delta vectors, live, attenuated delta viruses and herpes viral vectors (e.g., U.S. Pat. No. 5,288,641), as well as vectors which are disclosed within U.S. Pat. No. 5,166,320. Other representative vectors include retroviral vectors (e.g., EP 0 415 731; WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218). General methods of using such vectors in gene therapy are well known in the art (see, for example, Larrick, J. W. and Burck, K. L., Gene Therapy Application of Molecular Biology, Elsevier Science Publishing Co., Inc., New York, N.Y., 1991 and Kreigler, M., Gene Transfer and Expression; A Laboratory Manual, W.H. Freeman and Company, New York, 1990).

Further provided by this invention are vectors having more than one nucleic acid molecule encoding a ribozyme of this invention, each molecule under the control of a separate eukaryotic promoter (or, an Internal Ribosome Entry Site or "IRES") or alternatively, under the control of single eukaryotic promoter. Representative examples of other nucleic acid molecules which may be delivered by the vectors of the present invention include therapeutic molecules such as interferon (e.g., alpha, beta or gamma), as well as a wide variety of other cytokines or growth factors, and facilitators which assist or aid ribozymes in cleaving a target sequence by unwinding or otherwise limiting secondary folding which might otherwise inhibit the ribozyme. These vectors provide the advantage of providing multi-functional therapy against Psoriasis, preferably with the various therapies working together in synergy.

Host prokaryotic and eukaryotic cells stably harboring the vectors described above also are provided by this invention. Suitable host cells include bacterial cells, rat cells, mouse cells, and human cells.

Delivery

Within certain aspects of the invention, ribozyme molecules, or nucleic acid molecules which encode the ribozyme, may be introduced into a host cell or administered to a patient utilizing a vehicle, or by various physical methods. Representative examples of such methods include transformation using calcium phosphate precipitation (Dubensky et al., PNAS 81:7529-7533, 1984), direct microinjection of such nucleic acid molecules into intact target cells (Acsadi et al., Nature 352:815-818, 1991), and electroporation whereby cells suspended in a conducting solution are subjected to an intense electric field in order to transiently polarize the membrane, allowing entry of the nucleic acid molecules. Other procedures include the use of nucleic acid molecules linked to an inactive adenovirus (Cotton et al., PNAS 89:6094, 1990), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989), microprojectile bombardment (Williams et al., PNAS 88:2726-2730, 1991), polycation compounds such as polylysine, receptor specific ligands, liposomes entrapping the nucleic acid molecules, spheroplast fusion whereby E. coli containing the nucleic acid molecules are stripped of their outer cell walls and fused to animal cells using polyethylene glycol, viral transduction, (Cline et al., Pharmac. Ther. 29:69, 1985; and Friedmann et al., Science 244:1275, 1989), and DNA ligand (Wu et al, J. of Biol. Chem. 264:16985-16987, 1989). In one embodiment, the ribozyme is introduced into the host cell using a liposome.

Within further embodiments of the invention, additional therapeutic molecules (e.g., interferon) or facilitators may be delivered utilizing the methods described herein. Such delivery may be either simultaneous to, or before or after the delivery of a ribozyme or vector expressing ribozymes.

Pharmaceutical Compositions

As noted above, pharmaceutical compositions (or "medicaments") also are provided by this invention. These compositions contain any of the above described ribozymes, DNA molecules, vectors or host cells, along with a pharmaceutically or physiologically acceptable carrier, excipient, or, diluent. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Particularly preferred carriers include cholesterols such as DOTAP: cholesterol.

Additional ingredients added to the compositions for the express purpose of inhibiting endogenous ribonucleases include reducing agents such as dithiothreitol, detergents such as sodium dodecyl sulfate, and other agents such as vanidyl nucleotides, aurin tricarbocylic acid, hydrogen peroxide and RNA decoys such as tRNA.

Pharmaceutical compositions of the present invention may also be prepared to contain, or express (e.g., if a vector), one or more additional therapeutic molecules (e.g., interferon) or facilitators.

The pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes, including for example, topically, intradermally, or intraocullarly. In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.

Pharmaceutical compositions of the present invention are useful for both diagnostic and therapeutic purposes.

Therapeutic Methods

Methods of treating proliferative diseases such as psoriasis or PDR are also provided by this invention. More specifically, within one aspect of the present invention proliferative diseases such as psoriasis or PDR may be treated by administering to a warm-blooded animal (e.g., a human) a therapeutically effective amount of ribozyme, and/or, nucleic acid molecule or vector which encodes the ribozyme.

Briefly, cells in response to injury or other insult typically follow at least one of two possible pathways: normal wound healing or exaggerated growth. For normal wound healing accelerated growth and maturation of cells occurs in order to allow healing of the wound as soon as possible.

Proliferative diseases such as psoriasis, scarring and PDR are similar in many respects to the process of wound healing. Namely, cells are created and migrate in a relatively short period of time. However, if the proliferation is too rapid or the signals to proliferate continue for too long a period of time, the cells build up to form thickened lesions. This growth is supported by new blood vessels, as well as the infiltration of a variety of lymphocytes which produce a wide variety of growth factors (that further increases the proliferation of the cells). These cells can produce tissue degrading enzymes which cause the destruction of surrounding tissue. In some cases, the thickened lesions contract, distorting the surface of the skin or retina. The result of the exaggerated growth is the clinical symptoms associated with the proliferative disease.

The present invention provides the treatment of proliferative skin and eye diseases by contacting desired cells with an effective amount of ribozyme of this invention or, alternatively, by transducing the cell with an effective amount of vector having a nucleic acid molecule encoding the ribozyme. A suitable "therapeutically effective amount" will depend on the nature and extent of diseased tissue being treated, or, if a medical procedure is contemplated in which abnormal proliferation can be expected, prevented. Such "therapeutically effective amounts" can be readily determined by those of skill in the art using well known methodology, and suitable animal models (e.g. a rat, rabbit, or porcine model), or, based upon clinical trials. As utilized herein, a patient is deemed to be "treated" if the proliferative skin or eye disease is reversed or inhibited in a patient in a quantifiable manner. Further, a therapeutically effective amount or regimen of treatment should result in: (1) decrease in the frequency, severity, or, duration of clinical symptoms (e.g., inflammation, thickening of the tissue, contraction, scaling, "burning", or itching); (2) increase of time in the period of remission; (3) a change in pathological symptoms (e.g., inhibition of keratinocyte, fibroblast, glia, or retinal pigment epithelium proliferation); (4) prevention of retinal detachment; and/or (5) prevention of visual impairment.

When exogenously delivering the ribozyme, the RNA molecule can be embedded within a stable RNA molecule or in another form of protective environment, such as a liposome. Alternatively, the RNA can be embedded within RNase-resistant DNA counterparts. Cellular uptake of the exogenous ribozyme can be enhanced by attaching chemical groups to the DNA ends, such as cholesteryl moieties (Letsinger et al., P.N.A.S., U.S.A., 1989).

In another aspect of the invention, the target cell is transduced under conditions favoring insertion of the vector into the target cell and stable expression of the nucleic acid encoding the ribozyme. The target cell can include but is not limited to cells found in the skin basal level, dermis, or epidermis; the vitreous of the eye, and the retinal and pigment epithelium layers of the eye.

In addition, the ribozyme, ribozyme gene or vector may be readily incorporated into a biodegradable polymer, sphere, pleuroinc gel, or the like to aid incorporation into cells.

The ribozyme (or nucleic acid molecule or vector encoding the ribozyme) can be administered in any manner sufficient to achieve the above therapeutic results, but preferred methods include topical and systemic administration. Patients with localized disease can be administered ribozymes (or a nucleic acid molecule or vector) in a topical cream, ointment or emollient applied directly to skin lesions. For example, a topical cream containing 100 milligrams ribozymes by weight can be administered depending upon severity of the disease and the patient's response to treatment. In a preferred embodiment, a topical preparation containing ribozymes 100 milligrams by weight would be administered to psoriatic lesions. Alternatively, direct intracutaneous or intraocular injection of ribozymes in a suitable pharmaceutical vehicle can be used for the management of individual lesions.

The ribozyme may be formulated along with a liquid (e.g., DOTAP: cholesterol). In addition, the ribozyme may be formulated with ribonuclease inhibitors, including, but not limited to, reducing agent (e.g., dithiothreitol), detergent (e.g., sodium dodecyl sulfate), vanidyl nucleotides, aurin tricarboxcylic acid, hydrogen peroxide, and RNA decoys (e.g., tRNAs).

Claim 1 of 28 Claims

We claim:

1. A method of treating a proliferative eye disease, comprising administering locally to a patient a therapeutically effective amount of a ribozyme which cleaves RNA encoding a cyclin PCNA, such that said proliferative eye disease is treated, said RNA comprising SEQ ID NO: 4145, wherein the binding arms of said ribozyme bind to SEQ ID NO:4145.

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