The present invention provides methods and compositions for attenuating expression of a target gene
[0001] This application claims the benefit of priority from U.S. Provisional Application Nos. 60/336,314, filed November 2, 2001; 60/337,304, filed November 5, 2001; and 60/418,909, filed October 15, 2002, the specifications of each of which are incorporated by reference herein in their entirety.
[0002] The structure and biological behavior of a cell is determined by the pattern of gene expression within that cell at a given time. Perturbations of gene expression have long been acknowledged to account for a vast number of diseases including, numerous forms of cancer, vascular diseases, neuronal and endocrine diseases. Abnormal expression patterns, in form of amplification, deletion, gene rearrangements, and loss or gain of function mutations, are now known to lead to aberrant behavior of a disease cell. Aberrant gene expression has also been noted as a defense mechanism of certain organisms to ward off the threat of pathogens.
[0003] One of the major challenges of medicine has been to regulate the expression of targeted genes that are implicated in a wide diversity of physiological responses. While over-expression of an exogenously introduced transgene in a eukaryotic cell is relatively straightforward, targeted inhibition of specific genes has been more difficult to achieve. Traditional approaches for suppressing gene expression, including site-directed gene disruption, antisense RNA or co-suppress or injection, require complex genetic manipulations or heavy dosages of suppressors that often exceeds the toxicity tolerance level of the host cell.
[0004] RNA interference (RNAi) is a phenomenon describing double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Initial attempts to harness this phenomenon for experimental manipulation of mammalian cells were foiled by a robust and nonspecific antiviral defense mechanism activated in response to long dsRNA molecules. Gil et al. Apoptosis 2000, 5:107-114. The field was significantly advanced upon the demonstration that synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without invoking generic antiviral defense mechanisms. Elbashir et al. Nature 2001, 411:494-498; Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747. As a result, small-interfering RNAs (siRNAs) have become powerful tools to dissect gene function. The chemical synthesis of small RNAs is one avenue that has produced promising results. Numerous groups have also sought the development of DNA-based vectors capable of generating such siRNA within cells. Several groups have recently attained this goal and published similar strategies that, in general, involve transcription of short hairpin (sh)RNAs that are efficiently processed to form siRNAs within cells. Paddison et al. PNAS 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553. These reports describe methods to generate siRNAs capable of specifically targeting numerous endogenously and exogenously expressed genes.
[0005] One aspect of the present invention provides a stable respiratory formulation comprising RNAi constructs formulated for pulmonary or nasal delivery of a therapeutically effective amount of said RNAi constructs to the lungs of a patient. In certain embodiments, the RNAi constructs are formulated as microparticles having an average diameter less than 20 microns, and more preferably, having an average diameter of 0.5 to 10 microns. In certain embodiments, the microparticles are formed from biodegradable polymers. In certain embodiments, the microparticles are formed from one or more polymers selected from the group consisting of polysaccharides, diketopiperazines, poly(hydroxy acids), polyanhydrides, polyesters, polyamides, polycarbonates, polyalkylenes, poly vinyl compounds, polysiloxanes, polymers of acrylic and methacrylic acids, polyurethanes, celluloses, poly(butic acid), poly(valeric acid), and poly(lactide-co-caprolactone), or co-polymers thereof.
[0006] In certain embodiments, the microparticles are formed by solvent evaporation, spray drying, solvent extraction or hot melt encapsulation; while in other embodiments, the microparticles are in dry or lyophilized form. In other embodiments, the RNAi constructs are formulated in liposomes.
[0007] In still other embodiments, the RNAi constructs are formulated as supramolecular complexes including a multi-dimensional polymer network. Preferably, the supramolecular complexes are formed from cationic polymers such as poly(L)lysine (PLL), polyethylenimine (PEI), β-cyclodextrin containing polymers (βCD-polymers) or co-polymers thereof. More preferably, the supramolecular complexes are formed from cyclodextrin-modified polymers, for example, the cyclodextrin-modified poly(ethylenimine) having a structure of the formula:
[0008] R represents, independently for each occurrence, H, lower alkyl, a cyclodextrin moiety, or
[0009] m, independently for each occurrence, represents an integer from 2-10,000, preferably from 10 to 5,000, or from 100 to 1,000.
[0010] In certain embodiments, the respiratory formulation comprising RNAi constructs include a propellant.
[0011] In certain embodiments, the respiratory formulation is contained in a metered dose inhaler, a dry powder inhaler or an air-jet nebulizer. In a preferred embodiment, the RNAi construct is formulated in an amount to provide a therapeutically effective amount in one to ten meter doses.
[0012] Another aspect of the invention provides a metered dose aerosol dispenser containing an aerosol pharmaceutical composition for pulmonary or nasal delivery comprising a respirable formulation of RNAi constructs.
[0013] Yet another aspect of the invention provides a method for affecting systemic administration of an RNAi construct comprising administering to a patient, by way of pulmonary administration, a respirable formulation of RNAi constructs which is taken up in an amount in the deep lung to deliver a systemic dose of said RNAi construct.
[0014] Still another aspect of the invention provides a pharmaceutical preparation comprising at least one RNAi construct formulated for pulmonary or nasal delivery, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable salts, ester, and salts of such esters. In certain preferred embodiments, the present invention provides a pharmaceutical package comprising the pharmaceutical preparation which includes at least one RNAi construct formulated for pulmonary or nasal delivery and a pharmaceutically acceptable carrier, in association with instructions (written and/or pictorial) for administering the preparation to a human patient.
[0015] Another aspect of the present invention provides a composition comprising one or more RNAi constructs formulated in a supramolecular complex and in an amount sufficient to attenuate expression of a target gene in treated cells through an RNA interference mechanism. For example, the RNAi construct is an small-interfering RNA (siRNA), preferably being 19-30 base pairs in length. Alternatively, the RNAi construct is an expression vector having a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA in the treated cells. Optionally, the RNAi construct is a hairpin RNA which is processed to an siRNA in said treated cells. In certain embodiments, the composition is administered for treatment of cells
[0016] In certain embodiments, the supramolecular complexes are aggregated into particles having an average diameter of between 20 and 500 nm, and more preferably, between 20 and 200 nm.
[0017] To further illustrate, the supramolecular complex can be a multi-dimensional polymer network including linear polymers or branched polymers. Exemplary polymers are cationic polymers such as poly(L)lysine (PLL), polyethylenimine (PEI), β-cyclodextrin containing polymers (βCD-polymers) or co-polymers thereof. In certain embodiments, the supramolecular complexes are formed from cyclodextrin-modified polymers, for example, the cyclodextrin-modified poly(ethylenimine) having a structure of the formula:
[0018] wherein
[0019] R represents, independently for each occurrence, H, lower alkyl, a cyclodextrin moiety, or
[0020] m, independently for each occurrence, represents an integer from 2-10,000, preferably from 10 to 5,000, or from 100 to 1,000.
[0021] Another aspect of the present invention provides a method for attenuating expression of a target gene of a cell
[0022] Yet another aspect of the invention provides a pharmaceutical preparation comprising at least one RNAi construct formulated in a supramolecular complex, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable salts, ester, and salts of such esters. In certain preferred embodiments, the present invention provides a pharmaceutical package comprising the pharmaceutical preparation which includes at least one RNAi construct formulated in a supramolecular complex and a pharmaceutically acceptable carrier, in association with instructions (written and/or pictorial) for administering the preparation to a human patient.
[0023] Another aspect of the present invention provides a coating for use on a surface of a medical device, comprising a polymer matrix having RNAi constructs dispersed therein, which RNAi constructs are eluted from the matrix when implanted at site in a patient"s body and alter the growth, survival or differentiation of cells in the vicinity of the implanted device. Exemplary medical devices include screws, plates, washers, sutures, prosthesis anchors, tacks, staples, electrical leads, valves, membranes, catheters, implantable vascular access ports, blood storage bags, blood tubings, central venous catheters, arterial catheters, vascular grafts, intraaortic balloon pumps, heart valves, cardiovascular sutures, artificial hearts, pacemakers, ventricular assist pumps, extracorporeal devices, blood filters, hemodialysis units, hemoperfasion units, plasmapheresis units, and filters adapted for deployment in a blood vessel. A certain preferred embodiment provides a coated stent.
[0024] In certain embodiments, the RNAi construct of the coating is an small-interfering RNA (siRNA), preferably being 19-30 base pairs in length. Alternatively, the RNAi construct is an expression vector having a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA in the treated cells. Optionally, the RNAi construct is a hairpin RNA which is processed to an siRNA in the treated cells.
[0025] To illustrate, the RNAi construct of the coating can be one that attenuates at least one target gene selected from cyclin dependent kinases, c-myb, c-myc, proliferating cell nuclear antigen (PCNA), transforming growth factor-beta (TGF-beta), and transcription factors nuclear factor kappaB (NF-κB), E2F, HER-2/neu, PKA, TGF-alpha, EGFR, TGF-beta, IGFIR, P12, MDM2, BRCA, Bcl-2, VEGF, MDR, ferritin, transferrin receptor, IRE, C-fos, HSP27, C-raf, and metallothionein genes.
[0026] In certain preferred embodiments, the RNAi construct inhibits expression of a gene so as to attenuate proliferation and/or migration of smooth muscle cells.
[0027] Still another aspect of the present invention provides a method for coating a medical device with one or more RNAi constructs, comprising:
[0028] a) formulating the RNAi construct for coating a surface of a device such that said RNAi constructs are eluted from the surface when the device is implanted at site in a patient"s body; and
[0029] b) coating the formulated RNAi construct on a medical device,wherein the medical device coated with the RNAi construct attenuates expression of one or more genes in cells in the vicinity of the implanted device.
[0030] Another aspect of the present invention provides a composition comprising one or more RNAi constructs formulated for percutaneous intrapericardial delivery to an animal. In one embodiment, the RNAi construct of the composition attenuates expression of a gene resulting in increased angiogenesis and/or reduced ischemic damage in and around a myocardial infarct. Optionally, the RNAi construct is systemically available and attenuates expression of one or more genes in cells distal to the pericardial space.
[0031] In certain embodiments, the RNAi construct of the composition is encapsulated or associated with liposomes. For example, the liposomes are cationic liposomes formed from cationic vesicle-forming lipids. Optionally, the liposomes of the composition have an average diameter of less than about 200 nm.
[0032] In other embodiments, the RNAi construct is formulated as supramolecular complexes including a multi-dimensional polymer network. Preferably, the polymers are cationic polymers such as poly(L)lysine (PLL), polyethylenimine (PEI), β-cyclodextrin containing polymers (βCD-polymers) or co-polymers thereof.
[0033] In certain embodiments, the supramolecular complexes of the composition are formed from cyclodextrin-modified polymers, for example, the cyclodextrin-modified poly(ethylenimine) having a structure of the formula:
[0034] wherein
[0035] R represents, independently for each occurrence, H, lower alkyl, a cyclodextrin moiety, or
[0036]
[0037] m, independently for each occurrence, represents an integer from 2-10,000, preferably from 10 to 5,000, or from 100 to 1,000.
[0038] In certain embodiments, the RNAi construct of the composition is an small-interfering RNA (siRNA), preferably being 19-30 base pairs in length. Alternatively, the RNAi construct is an expression vector having a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA in the treated cells. Optionally, the RNAi construct is a hairpin RNA which is processed to an siRNA in the treated cells. In a preferred embodiment, the animal of the composition is a human.
[0039] In one embodiment, the present invention provides a pharmaceutical preparation comprising at least one RNAi construct formulated for percutaneous intrapericardial delivery, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable salts, ester, and salts of such esters. In certain preferred embodiments, the present invention provides a pharmaceutical package comprising the pharmaceutical preparation which includes at least one RNAi formulated for percutaneous intrapericardial delivery and a pharmaceutically acceptable carrier, in association with instructions (written and/or pictorial) for administering the preparation to a human patient.
[0040] Still another aspect of the present invention provides a method for percutaneous intrapericardial delivery of one or more RNAi constructs
[0041] Another aspect of the present invention provides a composition comprising one or more RNAi constructs formulated in liposomes for attenuating expression of a target gene of cells in vivo through an RNA interference mechanism. In certain preferred embodiments, the RNAi construct is an small-interfering RNA (siRNA), preferably being 19-30 base pairs in length. Alternatively, the RNAi construct is an expression vector having a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA in the treated cells. Optionally, the RNAi construct is a hairpin RNA which is processed to an siRNA in the treated cells. Preferably, the cell is a mammalian cell, such as a human cell.
[0042] In certain embodiments, the liposomes of the composition are cationic liposomes including cationic vesicle-forming lipids. In other embodiments, the liposomes have an average diameter of less than about 200 nm.
[0043] Still another aspect of the present invention provides a method for attenuating expression of a target gene of cells of a patient, comprising administering RNAi constructs formulated in liposomes and in an amount sufficient to attenuate expression of a target gene through an RNA interference mechanism, so as to thereby alter the growth, survival or differentiation of said cells. In certain preferred embodiments, the RNAi construct of the method is an small-interfering RNA (siRNA), preferably being 19-30 base pairs in length. Alternatively, the RNAi construct is an expression vector having a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA in the treated cells. Optionally, the RNAi construct is a hairpin RNA which is processed to an siRNA in the treated cells. Preferably, the cell the method is a mammalian cell, such as a human cell.
[0044] In certain embodiments, the liposomes of the method are cationic liposomes including cationic vesicle-forming lipids. In other embodiments, the liposomes have an average diameter of less than about 200 nm.
[0045] In certain embodiments, the present invention provides a pharmaceutical preparation comprising at least one RNAi construct formulated in liposomes, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable salts, ester, and salts of such esters. In certain preferred embodiments, the present invention provides a pharmaceutical package comprising the pharmaceutical preparation which includes at least one RNAi formulated in liposomes and a pharmaceutically acceptable carrier, in association with instructions (written and/or pictorial) for administering the preparation to a human patient.
[0046] Another aspect of the present invention provides a composition comprising one or more RNAi constructs formulated for electroporation into cells in vivo. For example, the RNAi construct is formulated in supramolecular complexes or in liposomes. In certain embodiments, the cells are epithelial cells or muscle cells.
[0047] Still another aspect of the present invention provides a method for delivering one or more RNAi constructs to a patient by electroporation, comprising administering the RNAi construct of sufficient amount to an animal through electroporation, wherein the RNAi construct attenuates expression of a target gene in cells of the patient. For example, the RNAi construct of the method is formulated in supramolecular complexes or in liposomes. In certain embodiments, the cells of the method are epithelial cells or muscle cells.
[0048] In one embodiment, the present invention provides a pharmaceutical preparation comprising at least one RNAi construct formulated for electroporation into cells, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable salts, ester, and salts of such esters. In certain preferred embodiments, the present invention provides a pharmaceutical package comprising the pharmaceutical preparation which includes at least one RNAi formulated for electroporation into cells and a pharmaceutically acceptable carrier, in association with instructions (written and/or pictorial) for administering the preparation to a human patient.
[0049] Another aspect of the present invention provides a composition comprising one or more formulated RNAi constructs for inhibiting unwanted cell growth in vivo, wherein, through an RNA interference mechanism, the RNAi construct reduces expression of a target gene essential to mitosis of a cell and/or which is essential to preventing apoptosis of said cell.
[0050] In certain preferred embodiments, the RNAi construct of the method is an small-interfering RNA (siRNA), preferably being 19-30 base pairs in length. Alternatively, the RNAi construct is an expression vector having a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA in the treated cells. For example, the expression vector is selected from an episomal expression vector, an integrative expression vector, and a viral expression vector. In another preferred embodiment, the RNAi construct is a hairpin RNA which is processed to an siRNA in the treated cells.
[0051] In certain embodiments, the RNAi construct of the composition inhibits proliferation of the cell. Alternatively, the RNAi construct promotes apoptosis of the cell.
[0052] Exemplary RNAi constructs inhibit expression of a target gene that is an oncogene, such as c-myc, c-myb, mdm2, PKA-I, Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent kinases, telomerase, PDGF/sis, erb-B, fos, jun, mos, src or the Bcr/Abl fusion gene.
[0053] In certain embodiments, the RNAi construct is used for the treatment of transformed cells, e.g., to inhibit or attenuate hyperplastic cell growth, and may be part of a treatment for cancer as such.
[0054] In other embodiments, the RNAi construct is used for inhibiting activation of lymphocytes, including treatment or prophylaxis of immune-mediated inflammatory disorders.
[0055] In still other embodiments, the RNAi construct is used for inhibiting proliferation of smooth muscle cells, including treatment or prophylaxis of restenosis.
[0056] In yet other embodiments, the RNAi construct is used for inhibiting proliferation of epithelial cells (e.g., as a component of cosmetic preparations).
[0057] In certain embodiments, the RNAi construct of the composition is formulated in a supramolecular complex. Optionally, the supramolecular complex comprises at least one polymer, for example, a cyclodextrin containing polymer. Alternatively, the RNAi construct is encapsulated or associated with a liposome, for example, a cationic liposome formed of a cationic vesicle-forming lipid. Optionally, the liposome complexed with the RNAi construct has a substantially homogeneous size of typically less than about 200 nm.
[0058] In a preferred embodiment, the animal is a human patient.
[0059] Still another aspect of the present invention provides a method for inhibiting unwanted cell growth in vivo, comprising administering to an animal a formulated RNAi construct of sufficient amount, wherein, through an RNA interference mechanism, the RNAi construct reduces expression of a target gene essential to mitosis of a cell and/or which is essential to preventing apoptosis of said cell.
[0060] In certain preferred embodiments, the RNAi construct of the method is an small-interfering RNA (siRNA), preferably being 19-30 base pairs in length. Alternatively, the RNAi construct is an expression vector having a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA in the treated cells. For example, the expression vector is selected from an episomal expression vector, an integrative expression vector, and a viral expression vector. In another preferred embodiment, the RNAi construct is a hairpin RNA which is processed to an siRNA in the treated cells.
[0061] In certain embodiments, the RNAi construct of the composition inhibits proliferation of the cell. Alternatively, the RNAi construct promotes apoptosis of the cell.
[0062] In certain preferred embodiment, the target gene is an oncogene, such as c-myc, c-myb, mdm2, PKA-I, Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent kinases, telomerase, PDGF/sis, erb-B, fos, jun, mos, src or the Bcr/Abl fusion gene. In certain embodiments, the cell is a transformed cell so that the RNAi construct is used for the treatment of hyperplastic cell growth, including treatment of a cancer. In other embodiments, the RNAi construct is used for inhibiting activation of lymphocytes, including treatment or prophylaxis of immune-mediated inflammatory disorders. In still other embodiments, the RNAi construct is used for inhibiting proliferation of smooth muscle cells, including treatment or prophylaxis of restenosis. In yet other embodiments, the RNAi construct is used for inhibiting proliferation of epithelial cells (e.g., as a component of cosmetic preparations).
[0063] In other embodiments, the RNAi construct of the method is formulated in a supramolecular complex. Optionally, the supramolecular complex comprises at least one polymer, for example, a cyclodextrin containing polymer.
[0064] Alternatively, the RNAi construct is encapsulated or associated with a liposome, for example, a cationic liposome formed of a cationic vesicle-forming lipid. Optionally, the liposome complexed with the RNAi construct has a substantially homogeneous size of typically less than about 200 nm.
[0065] In a preferred embodiment, the animal of the method is a human.
[0066] Another aspect of the invention provides a pharmaceutical preparation comprising at least one RNAi construct formulated for inhibiting unwanted cell growth, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable salts, ester, and salts of such esters. In certain preferred embodiments, the present invention provides a pharmaceutical package comprising the pharmaceutical preparation which includes at least one RNAi formulated for inhibiting unwanted cell growth and a pharmaceutically acceptable carrier, in association with instructions (written and/or pictorial) for administering the preparation to a human patient. In another embodiment, the present invention provides a cosmetic preparation comprising at least one RNAi construct formulated for inhibiting epithelial cell growth or differentiation.
[0067] Still another aspect of the present invention provides a method for inducing cell death, comprising administering to target cells in vivo an double stranded RNA, or an expression vector capable of transcribing a double stranded RNA, of sufficient length to activate a PKR response in the target cells, which double stranded RNA is formulated as part of a supramolecular complex.
[0068] In certain preferred embodiments, the double stranded RNA is more than 35 basepairs in length, and even more preferably more than 75, 100, 200 or even 400 basepairs. In certain embodiments, the target cells are mammalian cells, including transformed cells. In certain embodiments, the supramolecular complex is a multi-dimensional polymer network including linear polymers or branched polymers. Preferably, the supramolecular complex is formed from cationic polymers, such as poly(L)lysine (PLL), polyethylenimine (PEI), β-cyclodextrin containing polymers (βCD-polymers), and co-polymers thereof. 208.
[0069] In certain embodiments, the supramolecular complex is formed from cyclodextrin-modified polymers, including cyclodextrin-modified poly(ethylenimine) having a structure of the formula:
[0070] wherein
[0071] R represents, independently for each occurrence, H, lower alkyl, a cyclodextrin moiety, or
[0072]
[0073] m, independently for each occurrence, represents an integer from 2-10,000, preferably from 10 to 5,000, or from 100 to 1,000.
[0074] Still another aspect of the present invention provides a method of conducting a pharmaceutical business comprising:
[0075] a). identifying an RNAi construct which inhibits proliferation of target cells in vivo and reduces the effects of a disorder involving unwanted proliferation of the target cells;
[0076] b). conducting therapeutic profiling of the RNAi construct identified in step (a) for efficacy and toxicity in animals; and
[0077] c). formulating a pharmaceutical preparation including one or more RNAi constructs identified in step (b) as having an acceptable therapeutic profile.
[0078] Preferably, the method of conducting a pharmaceutical business includes an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and (optionally) establishing a sales group for marketing the pharmaceutical preparation.
[0079] Yet still another aspect of the present invention provides a method of conducting a pharmaceutical business comprising:
[0080] a). identifying an RNAi construct which inhibits proliferation of target cells in vivo and reduces the effects of a disorder involving unwanted proliferation of the target cells;
[0081] b). (optionally) conducting therapeutic profiling of the RNAi construct identified in step (a) for efficacy and toxicity in animals; and
[0082] c). licensing, to a third party, the rights for further development of the RNAi construct.
[0083]
[0084]
[0085] The present invention provides methods and compositions for attenuating expression of a target gene
[0086] One aspect of the invention relates to the use of RNAi constructs to attenuate expression of proliferation-regulating genes (including apoptosis-inhibiting genes). Such embodiments can be used as part of a therapeutic or cosmetic treatment program to inhibit, or at least reduce, unwanted growth of cells
[0087] Another aspect of invention relates to formulations of RNAi constructs for pulmonary administration, e.g., respirable RNAi constructs. Such formulations can be used for local or systemic delivery of RNAi constructs and constitute a convenient method for administration of RNAi constructs for any of a variety of indications, e.g., not limited to treating proliferative disorders. Merely to illustrate, certain RNAi compositions of the subject invention can be used to knockdown expression of vasoconstrictors, or reduce receptor levels of the vasoconstrictors, to reduce blood pressure in patients suffering from systemic and pulmonary hypertension.
[0088] Yet another aspect of the invention relates to methods of treating patients with RNAi constructs through percutaneous intrapericardial drug delivery in which the pericardial space is effectively used as a delivery reservoir for RNAi constructs. While useful in systemic delivery of RNAi constructs, it is contemplated that such techniques can be especially useful for local delivery to the heart and surrounding vasculature.
[0089] For instance, in the treatment of myocardial infarction, the invention provides a method for administering angiogenic RNAi constructs to promote angiogenesis and thereby promote recovery and/or prevent further damage to the tissue in an around the infarct. For instance, the subject method can be used to deliver an RNAi construct which inhibits expression of a protein that negatively regulates the activity of the NF-κB transcription factor, such as by inhibiting expressing of NF-κB repressor IκB, or any other cellular factor which reduces basic fibroblast growth factor-induced angiogenesis
[0090] Intrapericardial drug delivery can also be used for delivery of RNAi constructs which reduce proliferation and/or migration smooth muscle cells and thereby may be useful in treating neointimal hyperplasia, such as restenosis, artherosclerosis and the like. Merely to illustrate, inhibition of neointimal hyperplasia can be achieved by administration of RNAi constructs for attenuating gene expression of c-myb, c-myc, proliferating cell nuclear antigen (PCNA), transforming growth factor-beta (TGF-beta), and transcription factors such as nuclear factor kappaB (NF-κB) and E2F. In addition to intrapericardial drug delivery for reducing neointimal hyperplasia, the present invention also specifically contemplates the delivery of RNAi constructs "on stent", either by directly coating at least a portion of the stent with RNAi constructs, or through a polymeric coating from which the RNAi constructs are released.
[0091] Another aspect of the invention relates to coated medical devices. For instance, in certain embodiments, the subject invention provides a medical device having a coating adhered to at least one surface, wherein the coating includes the subject polymer matrix and an RNAi construct. Such coatings can be applied to surgical implements such as screws, plates, washers, sutures, prosthesis anchors, tacks, staples, electrical leads, valves, membranes. The devices can be catheters, implantable vascular access ports, blood storage bags, blood tubing, central venous catheters, arterial catheters, vascular grafts, intraaortic balloon pumps, heart valves, cardiovascular sutures, artificial hearts, a pacemaker, ventricular assist pumps, extracorporeal devices, blood filters, hemodialysis units, hemoperfasion units, plasmapheresis units, and filters adapted for deployment in a blood vessel.
[0092] Still another aspect of the invention provides compositions of RNAi constructs suitable for electroporation into cells
[0093] Another aspect of the invention, RNAi constructs are formulated in a supramolecular complex, and are suitable for use as a pharmaceutical agent, e.g., substantially free of pyrogenic agents. For instance, the supramolecular complex can be formed from a multi-dimensional polymer network comprising a linear polyethyleneimine or a linear cyclodextrin-containing polymer and a branched polyethyleneimine or branched cyclodextrin polymer. In certain preferred embodiments, the expression constructs are formulated with cyclodextrins, e.g., such as a cyclodextrin cellular delivery system.
[0094] Yet another aspect of the invention relates to the use of long double stranded RNA to activate the sequence independent dsRNA response in certain cells, e.g., to activate dsRNA-dependent protein kinase PKR. PKR-mediated response to long dsRNA (e.g., dsRNA greater than 35 basepairs, and even more preferably greater than 39, 50, 75, 100 or even 200 basepairs) is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. The subject compositions described herein for carrying out sequence-dependent RNA interference can be readily adapted to deliver long dsRNA (e.g., dsRNA molecule of sufficient length to activate PKR and induce cell death, such as in the range of 40-1000 basepairs, preferably 100-800 basepairs, and even more preferably 200-500 basepairs) to cells for which it is desired that cell death occur. In one embodiment, the subject long dsRNA formulations can be used to kill cancer cells.
[0095] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
[0096] As used herein the term "animal"refers to mammals, preferably mammals such as humans. Likewise, a "patient"or "subject" to be treated by the method of the invention can mean either a human or non-human animal.
[0097] The terms "apoptosis"or "programmed cell death,"refers to the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Apoptosis, is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise ("cellular suicide"). It is most often found during normal cell turnover and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system and endocrine-dependent tissue atrophy. Cells undergoing apoptosis show characteristic morphological and biochemical features. These features include chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria and nuclear material.
[0098] A "disease-associated" or "disease-causing" gene refers to any gene the expression of which is essential to or substantially contributes an unwanted cellular phenotype. It may be a gene that becomes expressed at an abnormally high level; it maybe a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at normal or abnormal level.
[0099] As used herein, the term "dsRNA" refers to siRNA molecules, or other RNA molecules including a double stranded feature and able to be processed to siRNA in cells, such as hairpin RNA moieties.
[0100] Likewise, the term "encodes," unless evident from its context, will be meant to include DNA sequences that encode a polypeptide, as the term is typically used, as well as DNA sequences that are transcribed into inhibitory antisense molecules.
[0101] The term "expression"with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein coding sequence results from transcription and translation of the coding sequence.
[0102] The "growth state"of a cell refers to the rate of proliferation of the cell and the state of differentiation of the cell.
[0103] As used herein, "immortalized cells" refers to cells that have been altered via chemical, genetic, and/or recombinant means such that the cells have the ability to grow through an indefinite number of divisions in culture.
[0104] "Inhibition of gene expression"refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene. "Specificity"refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radiolmmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
[0105] The term "loss-of-function,"as it refers to genes inhibited by the subject RNAi method, refers a diminishment in the level of expression of a gene when compared to the level in the absence of RNAi constructs.
[0106] As used herein, the phrase "mediates RNAi" refers to (indicates) the ability to distinguish which RNAs are to be degraded by the RNAi process, e.g., degradation occurs in a sequence-specific manner rather than by a sequence-independent dsRNA response, e.g., a PKR response.
[0107] The term "nasal delivery" refers to systemic delivery of RNAi constructs to a patient by inhalation through and into the nose.
[0108] As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
[0109] "Operably linked" when describing the relationship between two DNA regions simply means that they are functionally related to each other. For example, a promoter or other transcriptional regulatory sequence is operably linked to a coding sequence if it controls the transcription of the coding sequence.
[0110] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
[0111] As used herein, "proliferating" and "proliferation" refer to cells undergoing mitosis.
[0112] A "protein coding sequence" or a sequence that "encodes" a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide
[0113] The terms "pulmonary delivery" and "respiratory delivery" refer to systemic delivery of RNAi constructs to a patient by inhalation through the mouth and into the lungs.
[0114] By "recombinant virus" is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
[0115] As used herein, the term "RNAi construct" is a generic term used throughout the specification to include small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved
[0116] "RNAi expression vector" (also referred to herein as a "dsRNA-encoding plasmid") refers to a replicable nucleic acid constructs used to express (transcribe) RNA which produces siRNA moieties in the cell in which the construct is expressed. Such vectors include a transcriptional unit comprising an assembly of (1) genetic element(s) having a regulatory role in gene expression, for example, promoters, operators, or enhancers, operatively linked to (2) a "coding" sequence which is transcribed to produce a double-stranded RNA (two RNA moieties that anneal in the cell to form an siRNA, or a single hairpin RNA which can be processed to an siRNA), and (3) appropriate transcription initiation and termination sequences. The choice of promoter and other regulatory elements generally varies according to the intended host cell. In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
[0117] In the expression vectors, regulatory elements controlling transcription can be generally derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as retroviruses, adenoviruses, and the like, may be employed.
[0118] The term "small interfering RNAs"or "siRNAs"refers to nucleic acids around 19-30 nucleotides in length, and more preferably 21-23 nucleotides in length. The siRNAs are double-stranded, and may include short overhangs at each end. Preferably, the overhangs are 1-6 nucleotides in length at the 3" end. It is known in the art that the siRNAs can be chemically synthesized, or derive from a longer double-stranded RNA or a hairpin RNA. The siRNAs have significant sequence similarity to a target RNA so that the siRNAs can pair to the target RNA and result in sequence-specific degradation of the target RNA through an RNA interference mechanism. Optionally, the siRNA molecules comprise a 3' hydroxyl group.
[0119] The term "supramolecular complex"refers to a multi-dimensional polymer network formed with at least one polymer. The polymer molecule may be linear or branched, for example, poly(L)lysine (PLL), polyethylenimine (PEI), β-cyclodextrin containing polymers (βCD-polymers), and co-polymers thereof. In certain embodiments, the present invention relates to RNAi constructs formulated as supramolecular complexes.
[0120] "Transcriptional regulatory sequence" is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters and the like which induce or control transcription of coding sequences with which they are operably linked.
[0121] As used herein, the terms "transduction"and "transfection" are art recognized and mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. "Transformation," as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses an RNAi construct. A cell has been "stably transfected" with a nucleic acid construct when the nucleic acid construct is capable of being inherited by daughter cells.
[0122] As used herein, "transformed cells" refers to cells that have spontaneously converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. For purposes of this invention, the terms "transformed phenotype of malignant mammalian cells" and "transformed phenotype " are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals.
[0123] "Transient transfection" refers to cases where exogenous DNA does not integrate into the genome of a transfected cell, e.g., where episomal DNA is transcribed into mRNA and translated into protein.
[0124] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked. One type of vector is a genomic integrated vector, or "integrated vector," which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, i.e., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to that they are operatively linked are referred to herein as "expression vectors."In the present specification, "plasmid" and "vector" are used interchangeably unless otherwise clear from the context.
[0125] The RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the gene to be inhibited (i.e., the "target" gene). The double-stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. Thus, the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition.
[0126] Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating