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Title:  Reverse gene therapy

United States Patent:  6,852,704

Issued:  February 8, 2005

Inventors:  Levy; Robert J. (Merion Station, PA); Baldwin; Scott (West Chester, PA)

Assignee:  The Children's Hospital of Philadelphia (Philadelphia, PA)

Appl. No.:  487851

Filed:  January 19, 2000

Abstract

The invention relates to compositions and methods for reverse gene therapy, wherein a gene therapy vector encoding a gene product (e.g. a protein) which is usually only expressed in cells of an abnormal tissue is delivered to a cell of an animal afflicted with a disease or disorder to alleviate the disease or disorder. In one embodiment, a plasmid vector encoding HERG (A561V) protein is delivered to a cell of an animal afflicted with re-entrant atrial flutter-mediated cardiac arrhythmia.

Description of the Invention

BACKGROUND OF THE INVENTION

Re-entrant atrial flutter is a disease condition which affects many individuals. Electrophysiologic mapping techniques have lead to an enhanced understanding re-entrant atrial arrhythmias, and these advances have led to attempts to develop ablation procedures which destructively block conduction in myocardial regions involved in re-entry (e.g. a band of conductive tissue designated the crista terminalis which is involved in aberrant myocardial conduction associated with atrial flutter; Natale et al., 1996, Am. J. Cardiol. 78:1431-1433; Frame et al., 1996, Pacing Clin. Electrophysiol. 19:965-975; Cosio et al., 1996. Arch. Mal. Coeur Vaiss. 1:75-81; Cox et al., 1995, J. Thorac. Cardiovasc. Surg. 110:485-495, Cox et al., 1993. Ann. Thorac. Surg. 56:814-823; Cox et al., 1996, J. Thorac. Cardiovasc. Surg. 112:898-907; Arenal et al., 1999, Circulation 99:2771-2778).

BRIEF SUMMARY OF THE INVENTION

The invention relates to a method of alleviating a disease or disorder in an affected animal cell. The method comprises locally delivering to the cell a reverse gene therapy vector comprising a promoter operably linked with a nucleic acid encoding a therapeutic gene product which is usually only expressed in cells of an abnormal tissue that is not afflicted with the disease or disorder. Delivery of the reverse gene therapy vector to the affected cell and expression of the gene product therein alleviates the disease or disorder.

In one aspect of this method, the therapeutic gene product is a protein, such as one selected from the group consisting of a defective HERG protein, an apoptosis-inducing protein, transcription factor E2F1, tenascin C, bone morphogenic protein, a protein involved in synthesis of a glycosaminoglycan, a dominant negative mutant receptor protein, transcription factor NF-ATc, a mutant fibroblast growth factor receptor protein, and a degradation resistant collagen protein. Preferably, the protein is a defective HERG protein, such as HERG (A561V) protein.

In another aspect of the method, the reverse gene therapy vector is selected from the group consisting of naked DNA, a plasmid, a condensed nucleic acid, and a virus vector comprising a nucleic acid. The reverse gene therapy vector can, for example, be a virus vector (such as an adenovirus vector, a retrovirus vector, an adeno-associated virus vector, or a herpes virus vector), or a condensed nucleic acid. When a condensed nucleic acid reverse gene therapy vector is used, it can comprise a DNA molecule and a polycationic condensing agent.

In still another aspect of the method, the reverse gene therapy vector is a plasmid.

The polycationic condensing agent used in the method described herein can, for example, be selected from the group consisting of poly-L-lysine and Ca2+ ions. The promoter can be any promoter, including a constitutive promoter such as a CMV

Atrial fibrillation and atrial flutter are emerging as major clinical and public health problems for a number of reasons. The high incidence of atrial arrhythmias in the increasingly-aged population has resulted in the number of patients afflicted with atrial fibrillation or atrial flutter increasing into the millions (Prystowsky et al., 1996, Circulation 93:1262-1277; Anderson et al., 1996, Am. J. Cardiol. 78:17-21; Camm et al., 1996, Am. J. Cardiol. 78:3-11). In addition, atrial fibrillation and atrial flutter have been noted to occur very commonly following cardiac surgery, especially following coronary artery bypass surgery (Cox, 1993, Ann. Thorac. Surg. 56:405-409; Balaji et al., 1994, Am. J. Cardiol. 73:828-829; Balaji et al., 1994, J. Am. Coll. Cardiol. 23:1209-1215; Gandhi et al., 1996, Ann. Thorac. Surg. 61:1299-1309).

A number of mechanisms have been investigated to explain atrial arrhythmias, and are the basis for the conventional therapeutic approach. Re-entrant phenomena are thought to most often be the basis for atrial flutter (Gandhi et al., 1996, Ann. Thorac. Surg. 61:1666-1678; Frame et al., 1986, Circ. Res. 58:495-511; Frame et al., 1987, Circulation 5:1155-1175; Boyden et al., 1989, Circulation 79:406-416; Cosio et al., 1993, Lancet 341:1189-1193). Medications that slow atrial conduction or block down conduction through the AV-node have been useful for treatment of atrial arrhythmias (Waldo, 1994, Clin. Cardiol. 17:1121-1126, 1994; Wells et al., 1979. Circulation 60:665-673; Antman , 1996, Am. J. Cardiol. 78:67-72; Cochrane et al., 1996, Drug Ther. Bull. 34:41-45; Roden et al., 1996, Annu. Rev. Med. 47:135-48). Atrial fibrillation is believed often to result from a coalescence of multiple wavelets of impulse conduction (Moe, 1962, Arch. Int. Pharmacodyn. 1-2:183-188; Waldo, 1990. Circulation 81:1142-1143), and recent investigations have suggested that conditioned fibrillating atrium begets further atrial fibrillation (Salmon et al., 1985, Circulation 72(Suppl III):111-250; Morillo et al., 1995. Circulation 91:1588-1595; Wijffels et al., 1995. Circulation 92:1954-1968).

Gene Therapy

Gene therapy is generally understood to refer to techniques designed to deliver nucleic acids, including antisense DNA and RNA, ribozymes, viral genome fragments and functionally active therapeutic genes into targeted cells (Culver, 1994. Gene Therapy: A Handbook for Physicians. Mary Ann Liebert, Inc., New York, N.Y.). Such nucleic acids can themselves be therapeutic, as for example antisense DNAs that inhibit mRNA translation, or they can encode, for example, therapeutic proteins that promote, inhibit, augment, or replace cellular functions.

Virus vectors are among the most efficient gene therapy vectors which have been demonstrated. However, virus vectors sometimes elicit an immune response in the gene therapy host, which can inhibit the therapeutic benefit provided by the vector. Furthermore, use of retrovirus vectors can result in integration of the nucleic acid of the vector into the genome of the host, potentially causing harmful mutations. `Naked` nucleic acid vectors, such as linear DNA vectors and plasmids, do not generally induce an immune response or integrate into the host genome, but are taken up and expressed by host cells less effectively than virus vectors.

Among the shortcomings of current gene therapy strategies, including both ex vivo and in vivo gene therapy methods, is a dearth of appropriate nucleic acids for delivery to diseased or otherwise abnormal cells. Gene therapy methods have typically involved delivery of either a nucleic acid which is or which encodes a normal (i.e. wild type) component of a cell of the type to which the nucleic acid is delivered, an antisense oligonucleotide which inhibits or prevents transcription or translation of a nucleic acid in the diseased or abnormal cells, or a ribozyme which specifically cleaves a nucleic acid in diseased or abnormal cells. Although these nucleic acids can be effective in certain instances, a serious need remains for additional nucleic acids which, when delivered to diseased or abnormal cells, alleviate, prevent, or reverse the disease or abnormality. Furthermore, a gene therapy method which exerts its physiological effects by a mechanism which differs from the mechanism employed in previous gene therapy methods would be beneficial.

The present invention relieves these needs by providing compositions and methods for gene therapy which differ from the gene therapy compositions and methods of the prior art. promoter or a tissue-specific promoter such as a cardiac tissue-specific promoter (e.g. the ANF promoter or the .alpha.-MyHC promoter).

The reverse gene therapy vector used in the method described herein can further comprise a pharmacological agent-sensitive enhancer, such as a phorbol ester-sensitive enhancer. The reverse gene therapy vector can also, or alternatively, further comprise a Cre-recombinase-sensitive site.

According to the method of the invention, the reverse gene therapy vector can be delivered to the cell in a sustained-release manner. Such delivery methods can, for example, comprise delivering the reverse gene therapy vector to the cell in a form selected from a particle comprising the vector, a microparticle comprising the vector, a nanoparticle comprising the vector, an implantable device having a surface coated with a matrix comprising the vector, or a bulk material comprising the vector. The implantable device can, for example, comprise an electrode located in close proximity to a myocardial tissue of the animal, such as right atrial myocardium.

In one embodiment of the method described herein, the cell is located outside the body of the animal. The cell can, for example, be a cultured cell, such as a cultured cell which is subsequently returned to the body of the animal from which the cell was obtained or is subsequently returned to the body of a second animal other than the animal from which the cell was obtained.

In another embodiment of the method described herein, the cell is located inside the body of the animal. For example, the cell can be located in a cardiac tissue of the animal, such as a myocardial cell (e.g. a right atrial myocardium cell or a cell of the crista terminalis). The animal can be one which is afflicted with re-entry atrial flutter, in which event the therapeutic gene product is preferably a defective HERG protein, such as HERG (A561V) protein. Also preferably, the protein is operably linked with a cardiac tissue-specific promoter, such as one selected from the group consisting of the ANF promoter and the .alpha.-MyHC promoter.

The invention also relates to a reverse gene therapy vector for alleviating a disease or disorder in an affected cell. The vector comprises a promoter operably linked with a nucleic acid encoding a therapeutic gene product which is normally only expressed in cells of an abnormal tissue that is not afflicted with the disease or disorder. Delivery of the vector to the affected cell and expression of the gene product therein alleviates the disease or disorder.

In one aspect, the therapeutic gene product is a protein, such as one selected from the group consisting of a defective HERG protein, an apoptosis-inducing protein, transcription factor E2F1, tenascin C, bone morphogenic protein, a protein involved in synthesis of a glycosaminoglycan, a dominant negative mutant receptor protein, transcription factor NF-ATc, and a degradation resistant collagen protein. When the protein is a defective HERG protein, it is preferably HERG (A561V) protein.

In another aspect of the reverse gene therapy vector, the vector is selected from the group consisting of naked DNA, a plasmid, a condensed nucleic acid, and a virus vector comprising a nucleic acid. In one embodiment, the vector is a virus vector such as an adenovirus vector. In another embodiment, the vector is a condensed nucleic acid, such as one comprising a DNA molecule and a polycationic condensing agent. In still another embodiment, the gene therapy vector is a plasmid.

The polycationic condensing agent of the reverse gene therapy vector can, for example, be selected from the group consisting of poly-L-lysine and Ca2+ ions.

The promoter used in the reverse gene therapy vector can be substantially any promoter, including a constitutive promoter such as a CMV promoter or a tissue-specific promoter such as a cardiac tissue-specific promoter (e.g. the ANF promoter, the .alpha.-MyHC promoter, and the wild type HERG promoter).

The reverse gene therapy vector can further comprise a pharmacological agent-sensitive enhancer, such as a phorbol ester-sensitive enhancer. The reverse gene therapy vector can also, or alternatively, comprising a Cre-recombinase-sensitive site.

The invention also includes a particle, a microparticle, or a nanoparticle comprising the reverse gene therapy vector.

The invention further includes an implantable device comprising the reverse gene therapy vector, such as one having a surface coated with a matrix comprising the reverse gene therapy vector.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a new method of gene therapy herein designated `reverse` gene therapy. Traditional gene therapy methods involve using a gene vector to deliver a wild type or engineered gene or a promoter operably linked with a nucleic acid encoding a wild type or engineered protein or a wild type or engineered RNA molecule to an cell of an animal afflicted with a disease or disorder.

`Reverse` gene therapy, as described herein, refers to localized delivery of a gene therapy vector which comprises a nucleic acid to an affected cell of an animal afflicted with a disease or disorder. The nucleic acid encodes a therapeutic gene product which is usually only expressed in cells of an abnormal tissue which is not afflicted with the same disease or disorder. Such abnormal tissues include, for example, tissues afflicted with a different disease or disorder than the one being alleviated by reverse gene therapy. Because the therapeutic gene product is expressed in an abnormal tissue, expression of the therapeutic gene product in tissues other than the tissue afflicted with the disease or disorder being alleviated is generally considered by others to be undesirable (despite the `therapeutic` designation attached to such gene products in the present disclosure). Hence, it is necessary to minimize or prevent expression of the therapeutic gene product in normal tissues by delivering the gene therapy vector in a localized fashion, and preferably by expressing the therapeutic gene product in a tissue-specific manner. Also preferably, the gene therapy vector is administered in a sustained-release fashion in order to maximize and extend expression of the therapeutic gene product in the tissue afflicted with the disease or disorder being treated. The word "reverse" in reverse gene therapy emphasizes a nucleic acid construct which would be harmful if expressed in one physiological setting is delivered to a diseased physiological site in order to achieve the reverse (i.e. a beneficial) effect in a different setting.

The reverse gene therapy method is a method of alleviating a disease or disorder in an affected animal cell. This method comprises locally delivering to the cell a gene therapy vector. The gene therapy vector comprises a promoter operably linked with a nucleic acid encoding a therapeutic gene product which is usually only expressed in cells of an abnormal tissue that is not afflicted with the disease or disorder, such as cells of a tissue afflicted with a different disease or disorder. Delivery of the gene therapy vector to the affected cell and expression of the therapeutic gene product therein alleviates the disease or disorder in the cell. By alleviating the disease or disorder in individual affected cells of an animal afflicted with a disease or disorder, the symptoms of the disease or disorder are alleviated. In contrast with alleviation of symptoms effected by administration of non-nucleic acid-containing pharmaceutical agents, administration of the gene therapy vector described herein results in a longer period of relief from the symptoms. If the gene therapy vector comprises a virus vector which is capable of integrating its nucleic acid into the genome of the cell or into the genome of an organelle within the cell, very long term relief can be effected, possibly enduring for the length of the animal's life.

Preferred compositions and methods for reverse gene therapy which are described herein include compositions and methods for delivering a gene therapy vector to cardiac tissue in an animal afflicted with a cardiac disease or disorder such as cardiac arrhythmias. Localized delivery of pharmaceutical agents to cardiac tissue has been described by others (e.g. Labhasetwar et al., 1998, J. Cardiovasc. Pharmacol. 31:449-455; Labhasetwar et al., 1997, Adv. Drug Del. Rev. 24:109-120; Labhasetwar et al., 1997, Adv. Drug. Del. Rev. 24:63-85; Sintov et al., 1997, Int. J. Pharm. 146:55-62; Gottsauner-Wolf et al., 1997. Am. Heart J. 133:329-334; Humphrey et al., 1997, Adv. Drug Delivery Rev. 24: 87-108; Desai et al., 1997, Pharm. Res. 14:1568-1573: Song et al., 1997, J. Controlled Release 45:177-192).

Localized delivery of an agent such as a gene therapy vector advantageously delivers the agent only or primarily to a particular site, minimizes the amount of agent which needs to be delivered (i.e. by minimizing delivery to undesired sites), and minimizes undesirable effects caused by delivery of the agent systemically or to tissues located at a distance from the particular site. By way of example, enhanced efficacy of various anti-arrhythmic agents has been demonstrated when the agents were locally delivered, relative to the efficacy of the same agents delivered systemically (Labhasetwar et al., 1997, Adv. Drug Del. Rev. 24:109-120; Labhasetwar et al., 1997, Adv. Drug. Del. Rev. 24:63-85; Sintov et al., 1997, Int. J. Pharm. 146:55-62; Gottsauner-Wolf et al., 1997, Am. Heart J. 133:329-334; Humphrey et al., 1997, Adv. Drug Delivery Rev. 24:87-108; Desai et al., 1997, Pharm. Res. 14:1568-1573; Song et al., 1997, J. Controlled Release 45:177-192). Reduction of ventricular defibrillation thresholds has also been associated with local cardiac drug delivery (Song et al., 1997. J. Controlled Release 45:177-192).

A drawback of sustained-release drug delivery of a conventional pharmaceutical agent is the need to continuously resupply drug to the drug reservoir because of depletion or turnover of the drug. Sustained-release delivery of many anti-arrhythmics is further hindered by the relatively non-specific effect of such agents and by the fact that local delivery of such agents fails to change the nature of the underlying pro-arrhythmic myocardium. Thus, when delivery of anti-arrhythmic agent ceases, the myocardium remains pro-arrhythmic.

Traditional gene therapy methods have not been useful for treating pro-arrhythmic myocardium because of several factors. First, no reasonable candidate genes have been proposed for delivery to pro-arrhythmic myocardium. Second, delivery systems for localizing gene vector delivery to specific arrhythmogenic circuits within the heart have not been previously described. Third, numerous gene vectors suggested for gene therapy have exhibited complications relating to, among other things, systemic immunogenicity and toxicity. The present invention overcomes these shortcomings. As described herein, reverse gene therapy can be used to appropriately alter myocardial sites involved in mechanistic events leading to re-entrant arrhythmias because use of pathologic mutants of ion channel proteins defeats tachyarrhythmic conduction circuits and achieves, in essence, a "biotech ablation" of such arrhythmias. Perhaps because these mutant proteins are usually only expressed in cells of an abnormal tissue, their use to treat alleviate arrhythmias and other cardiac disease and disorders has not been contemplated by others.

The identity of the therapeutic gene product is not critical. This gene product need only be one which will alleviate the disease or disorder in the affected cells or tissues. When the disease or disorder is re-entry atrial flutter, the gene product can be any gene product that reduces myocardial conductivity in atrial tissue. Examples of such gene products include mutated ion channel proteins and their subunits. These proteins and normally-disease/disorder-associated mutant forms thereof, are described, for example in McDonald et al. (1997, Nature 388:289-292). Expression of such proteins/subunits is normally associated with a disease or disorder. However, when these proteins/subunits are expressed in atrial tissue in a subject afflicted with re-entry atrial flutter, conductivity of the tissue is reduced, and the atrial flutter is alleviated. An example of an ion channel protein is HERG.

HERG refers to the human ether agogo gene, which encodes a potassium channel rectifier protein that modulates myocardial K+ re-entrant current. HERG (A561V) refers to a point mutation (resulting in an alanine-to-valine substitution) in this protein, which is responsible for one of the forms of the Long QT Syndrome, a hereditary disorder associated with episodes of ventricular arrhythmias and a risk of sudden death (Labhasetwar et al., 1995. Proc. Natl. Acad. Sci. USA 92:2612-2616; Schwendeman et al., 1995, Pharm. Res. 12:790-795; Labhasetwar et al., 1995, Clin. Pharmacokinet. 29:1-5: Levy et al., 1995, J. Controlled Release 36:137-147; Gibson et al., 1995, In: Molecular Interventions and Local Drug Delivery in Cardiovascular Disease, Edelman, Ed., W. B. Saunders Co., Ltd., London, UK, pp. 327-352; Wood et al., 1995. In: Molecular Interventions and Local Drug Delivery in Cardiovascular Disease, Edelman. Ed., W. B. Saunders Co., Ltd., London, UK, pp. 399-471). The HERG gene resides on chromosome 7 (q35-36), and has a length of about 3.2 kilobases, cDNA encoding HERG (A561V) protein has been incorporated into a plasmid vector by others, and this plasmid was used to define the mechanism of its role in the Long QT Syndrome (Wood et al., 1995, In: Molecular Interventions and Local Drug, Delivery in Cardiovascular Disease, Edelman, Ed., W. B. Saunders Co., LTD, London, UK, pp. 399-471). Expression of HERG (A561V) in Xenopus oocytes depressed the tail current response to various test pulses of voltage amplitudes, which indicated that HERG (A561V) becomes associated with the cell membrane following introduction of exogenous genetic material (Sanguinetti et al., 1996, Proc. Natl. Acad. Sci. USA. 93:2208).

The HERG (A561V) gene encodes a defective potassium channel rectifier. Defective HERG (A561V) protein interacts with the wild type HERG potassium channel rectifier in a dominant negative manner, thereby inhibiting K- current through the HERG membrane protein. Expression of the defective HERG (A561V) protein in the cell membrane of cardiac myocytes results in prolonged myocardial conduction. Ibutilide, a short acting Class III anti-arrhythmic agent, also blocks cardiac potassium channel rectifier current and delays myocardial conduction. Ibutilide has been administered to patients to prevent re-entrant atrial flutter. Because both ibutilide and defective HERG (A561V) protein inhibit K- current through the HERG membrane protein, administration of defective HERG (A561V) protein to a patient afflicted with re-entrant atrial flutter using a reverse gene therapy method as described herein will relieve this condition. Prior to ethical use of this reverse gene therapy method on human patients, the method is tested using dogs. Dogs are utilized in these studies, because of the extensive prior work by the inventors and many others on dog models of cardiac arrhythmias and, in particular, atrial flutter (e.g. Kirshenbaum et al., 1996, Develop. Biol. 179:402-411; Cox et al., 1995, J. Thorac. Cardiovasc. Surg. 110:485-495). Dog myocardium is thus an art-recognized model of human myocardium, at least for the purposes of assessing the effectiveness of alleviating re-entrant atrial flutter.

Although the compositions and methods described herein focus on use of HERG (A561V), one or more of the other point mutations which have been described in the human ether agogo gene can be similarly used (e.g. Labhasetwar et al., 1995, Proc. Natl. Acad. Sci. USA 92:2612-2616; Schwendeman et al., 1995, Pharm. Res. 12:790-795; Labhasetwar et al., 1995, Clin. Pharmacokinet. 29:1-5; Levy et al., 1995, J. Controlled Release 36:137-147; Gibson et al., 1995, In: Molecular Interventions and Local Drug Delivery in Cardiovascular Disease, Edelman, Ed., W. B. Saunders Co., Ltd., London, UK, pp. 327-352). Alternatively, re-entrant circuit block can elicited by localized delivery and expression of the transcription factor, E2F1, which causes apoptosis in mature myocytes (Levy 1995, In: Molecular Interventions and Local Drug Delivery in Cardiovascular Disease, Edelman, Ed., London, UK: W. B. Saunders Co., Ltd.; Anderson et al., 1995, J. Biomed. Mater. Res. 29:1473-1475), thereby creating a devitalized region (by means of gene-induced apoptosis) within a re-entry loop.

Another example of a mutant HERG gene that is normally associated with an aberrant physiological condition (i.e. a disease or disorder) is the gene encoding the beta subunit of HERG. This gene encodes a protein that is designated MIRP (McDonald et al., 1997, Nature 388:289-292). MIRP mutants normally interfere with the physiological function of HERG, resulting in a disease condition. However, providing a MIRP mutant to atrial myocardium in a subject afflicted with re-entry atrial flutter decreases the conductivity of the atrial tissue, thereby alleviating the disorder.

The therapeutic gene product described herein is not limited to mutant ion channel proteins. The therapeutic gene product can be any gene product expression of which is associated with a first disease or disorder in an animal tissue, but which alleviates a different disease or disorder when it is expressed in an animal afflicted with a different disease or disorder. For example, many diseases and disorders can be alleviated by ablating particular cells or tissues. Thus, using the methods described herein, expression in those cells or tissues of a gene product that ablates those cells or tissues in a different disease or disorder leads to death of the cells/tissues. By way of example, a mutant fibroblast growth factor receptor protein is associated with induced apoptosis of smooth muscle cells in animal cells (Miyamoto et al., 1998, J. Cell. Physiol. 177:58-67). To continue the example involving re-entry atrial flutter, expression of this mutant receptor protein in atrial myocardial cells of an animal afflicted with re-entry atrial flutter induces apoptosis in those cells, ablating the conductive loop associated with the disorder. Thus, providing this mutant protein to these cells using the methods described herein alleviates the disorder.

Localization of delivery of an agent encoded by a nucleic acid can be enhanced by use of a tissue-specific or physiologically responsible promoter operably linked with the nucleic acid encoding the agent. Numerous tissue-specific and physiologically responsible promoters have been described. For example, tissue specific promoters and physiologically responsible promoters include the sm22alpha promoter, which specifically promotes expression of genes in arterial smooth muscle cells (Solway et al., 1995, J. Biol. Chem. 270:13460-13469) and the tenascin-C promoter, which specifically promotes expression of genes in proliferating cells in response to the presence of matrix metalloproteinase-modified collagens (Chiquet et al., 1996, Biochem. Cell Biol. 74:737-744: Copertino et al., 1997, Proc. Natl. Acad. Sci. USA 94:1846-1851).

A physiologic responsive promoter is a nucleotide sequence which regulates downstream DNA expression in response to a change in the regional physiology such as an alteration in the extracellular matrix (i.e. collagen breakdown or denaturation), an increase in regional temperature to the febrile range, or a response to a change in blood pressure or blood flow.

In the reverse gene therapy compositions and methods described herein for treatment of cardiac arrhythmias, the promoter is preferably a cardiac tissue-specific promoter, such as the .alpha.-myosin heavy chain promoter (.alpha.-MyHC; Anderson et al., 1995, Tissue Eng. 1:323-326; Villa et al., 1995. Circ. Res. 76:505-513) or the atrial natriuretic factor promoter (ANF; Guzman et al., 1996. Circulation 94:1441-1448). Of course, non-tissue-specific promoters (e.g. the wild type HERG promoter) and constitutive promoters (e.g. a cytomegalovirus {CMV} promoter) can be used in the gene therapy vector described herein.

Localized expression of a therapeutic gene product can be enhanced in a reverse gene therapy method by delivering a gene therapy vector having a nucleic acid which comprises a pharmacological agent-sensitive enhancer element in addition to the portion of the nucleic acid encoding the therapeutic gene product. A variety of such pharmacological agent-sensitive enhancer agents have been described, such as those which enhance gene expression in response to administration of a phorbol ester to a cell which comprises a nucleic acid having such an enhancer element (Desai et al., 1996, Pharm. Res. 13:1838-1845; Levy et al., 1996, Drug Delivery 3:137-142; Song et al., 1997, J. Controlled Release 43:197-212). Localized enhancement of expression of the therapeutic gene product can be effected by localized delivery of the gene therapy vector coupled with systemic delivery of the pharmacological agent corresponding to the enhancer element, by systemic delivery nf the gene therapy vector coupled with localized delivery of the pharmacological agent corresponding to the enhancer element, or, preferably, by localized delivery of both the gene therapy vector and the pharmacological agent corresponding to the enhancer element.

Expression of a gene product encoded by the gene therapy vector described herein can be rendered terminable by incorporating a Cre-recombinase sensitive site in the nucleic acid of the gene therapy vector, as described (Hammond et al., 1997, Analyt. Chem. 69:1192-1196). Expression of the gene product in a cell transformed using the gene therapy vector is terminated by delivering a second vector to the cell, wherein the second vector encodes Cre-recombinase.

In an alternative embodiment of the invention, the gene therapy vector encodes a protein which, when expressed in a cell, induces apoptosis of the cell. Such proteins include, for example the transcription factor E2F1 and transcription factors normally encoded by viruses (Levy, 1995, In: Molecular Interventions and Local Drug Delivery in Cardiovascular Disease, Edelman. Ed., London, UK: W. B. Saunders Co., Ltd.; Anderson et al., 1995. J. Biomed. Mater. Res. 29:1473-1475; Martin et al., 1995. Nature 375:691-694). Another example of such a protein is the mutant fibroblast growth factor receptor protein described above.

Other specific embodiments of the invention include the following:

Delivery of a gene therapy vector encoding a mutant tenascin C protein associated with a disease state to cardiac or coronary artery tissue, in order to limit or prevent progression or development of cardiac valve obstruction or coronary artery obstruction. Tenascin C normally organizes progressive deposition of extracellular matrix. In certain disease states, expression of mutant tenascin C proteins lead to repression of extracellular matrix production (Nakao et al., 1998, Am. J. Pathol. 152:1237-1245).

Delivery of a gene therapy vector encoding a bone morphogenic protein (BMP) under the transcriptional control of a mutant BMP promoter associated with a disease state to a bone fracture site or to a bone site at risk of fracture (e.g. bone non-union sites, sites at which reconstructive surgery has been performed, and cranio-facial sites). In certain disease states, mutant BMP promoters lead to overexpression of BMP (Kaplan et al., 1998, Biochem. Pharmacol. 55:373-382).

Delivery of a gene therapy vector comprising at least a portion of a mutant gene associated with one or more mucopolysaccharidoses to a glycosaminoglycan-(GAG-) deficient site or to a biomechanically compromised site (e.g. a joint, tendon, or heart valve) in the body of an animal. As is known, various mutant genes associated with one or more mucopolysaccharidoses result in overexpression of GAG in the affected tissue (Froissart et al., 1998, Clin. Gen. 53:362-368).

Delivery of a gene therapy vector encoding a mutant gene, expression of which mutant gene is associated with apoptosis in a disease state, to cells or tissue which contributes to a different disease state (e.g. delivery of an apoptosis-inducing gene to myocardium cells which form all or part of conduction pathway associated with arrhythmia). Numerous mutant genes are known, expression of which mutant gene is associated with apoptosis in a disease state (e.g. Nishina et al., 1997, Nature 385:350-353).

Delivery of a gene therapy vector encoding a mutant gene encoding a dominant negative mutant gene product associated with a disease state to cells or tissue which is affected by a disease state associated with the normal (i.e. non-mutant) form of the gene product. By way of example, dominant negative mutant variants of numerous cell-surface receptors are known, such as dominant negative mutants wherein one or more inoperative receptor subunits ablate the activity of a multi-subunit receptor (e.g. Kim et al., 1 998, J. Clin. Invest. 101:1821-1826).

Delivery of a gene therapy vector encoding therapeutic gene product which is usually only expressed in cells of an abnormal tissue to facilitate implantation of engineered tissue (e.g. cultured organ tissue) into an animal. For example, a vector comprising a disease-associated gene could be used to favorably modify a tissue prior to implantation of the tissue. By way of specific example, a gene that normally encodes a product which, when expressed, induces a skeletal defect (e.g. a gene described by Kaplan et al., 1998, Biochem. Pharmacol. 55:373-382) can be delivered to a tissue-engineered heart valve prior to implantation of the valve in a patient in order to prevent the valve from calcifying.

Delivery of a gene therapy vector encoding an uncontrollable mutant of the transcription factor NF-ATc to cardiac tissue of a post-natal individual to facilitate development of a cardiac valve. The role of transcription factor NF-ATc in abnormal cardiac valve formation has been described (Ranger et al., 1998, Nature 392:186-190).

Delivery of a gene therapy vector comprising a pressure- or flow-unresponsive mutant tenascin C gene (or cDNA) to cardiac tissue to retard or prevent cardiac valve obstruction. Such mutant tenascin C genes have been described (e.g. Huang et al., 1995, Nature 378:292-295).

Delivery of a gene therapy vector encoding a degradation resistant protein normally associated with a disease state to cells or tissue affected by a different disease state associated with the corresponding normal (i.e. degradation sensitive) form of the protein. For example, a gene therapy vector encoding a mutant collagen protein which is resistant to degradation by matrix metalloproteinase (MMP) can be delivered to a cell to block MMP cascade-integrin signaling (King et al., 1997, J. Biol. Chem. 272:28518-28522).

Delivery of a gene therapy vector comprising a gene having a deletion therein, relative to the wild type gene, wherein expression of the gene having the deletion is normally associated with a disease state, but when the gene therapy vector is delivered to cells or tissue affected by a different disease state, expression of the gene having the deletion alleviates or inhibits the different disease state. For example, chromosomal deletions such as the chromosome 22 deletions associated with cardiac defects (e.g. those described by Rauch et al., 1998. Am. J. Med. Gen. 78:322-331) can be used to inhibit heart valve calcification through by delivering vectors comprising antisense constructs corresponding to the deleted regions of chromosome 22. Delivery of such vectors to heart valve tissue suppresses differentiation of potentially calcifying cells in cardiac valves and blood vessels.

The Reverse Gene Therapy Vector

The invention includes a reverse gene therapy vector which is useful for alleviating a disease or disorder in a cell. This reverse gene therapy vector comprises a promoter operably linked with a nucleic acid encoding a therapeutic gene product which is normally only expressed in cells of an abnormal tissue that is not afflicted with the same disease or disorder. Delivery of the vector to the cell alleviates the disease or disorder.

The therapeutic gene product encoded by gene therapy vector described herein can, for example, be a protein, a ribozyme, an antisense RNA molecule, or another molecule which, when expressed in a normal cell, causes the normal cell to exhibit a symptom associated with a disease or disorder but which, when expressed in a cell to which the gene therapy vector is delivered, alleviates a symptom of a disease or disorder which affects the cell. Proteins which can be encoded by the gene therapy vector include defective HERG proteins, HERG (A561V) protein, apoptosis-inducing proteins, and transcription factor E2F1.

The reverse gene therapy vector can be substantially any nucleic acid vector which is now known or hereafter developed. Exemplary vectors include naked DNA vectors, plasmids, condensed nucleic acids, and virus vectors. In a preferred embodiment of the reverse gene therapy vector, the vector is a plasmid, and more preferably comprises both a plasmid and a condensing agent such as poly-L-lysine or Ca2+ ions. When the vector is a virus vector, the virus vector is preferably one of an adenovirus vector, a retrovirus vector, an adeno-associated virus vector, and a herpes virus vector.

Plasmid DNA transformation of mammalian cells results in plasmid DNA residing in the nucleus of the transfected cell, wherein the plasmid not incorporated into a chromosome. Transient episomal expression of plasmid DNA generally occurs following transformation (Dowty et al., 1995, Proc. Natl. Acad. Sci. USA 92:4572-4576; Wolff et al., 1996. Hum. Mol. Genet. 1:363-369; Fritz et al., 1996, Hum. Gene Ther. 7:1395-404). Plasmid transformation of cardiac and skeletal striated muscular tissue, either cardiac or skeletal, has been demonstrated following administration of naked DNA to such tissue, and expression of the DNA in the transformed cells has been observed to persist for months (Dowty et al., 1995. Proc. Natl. Acad. Sci. USA 92:4572-4576; Wolff et al., 1996, Hum. Mol. Genet. 1:363-369. Fritz et al., 1996, Hum. Gene Ther. 7:1395-404). Alternatively, a gene therapy vector, such as any of certain virus vectors, can be used, wherein the vector causes the nucleic acid carried thereby to be integrated into the host cell genome.

The gene therapy vector described herein is preferably administered to a cell or tissue of an animal in a sustained-release manner. Numerous methods have been described for effecting sustained release of a nucleic acid vector such as a gene therapy vector, and all known and hereafter-developed methods for achieving sustained release of a nucleic acid vector can be used in accordance with the compositions and methods described herein. The gene therapy vector is preferably DNA in the form of a plasmid, particularly condensed plasmid DNA incorporated into particles, microparticles, nanoparticles, a bulk material, or a coating present at a surface of an implantable device. Preferred nucleic acid vector compositions and methods of using them to administer a vector, such as the gene therapy vector described herein, are described in commonly-assigned U.S. patent applications having application Ser. Nos. 60/116,538; 60/116,405; and Ser. No. 09/234,011, each of which shares a common priority date with the present disclosure, and each of which is incorporated herein by reference.

When the gene therapy vector described herein comprises a gene therapy vector for delivering a therapeutic gene product to a cardiac tissue in order to alleviate a cardiac arrhythmia, the vector is preferably delivered to myocardial tissue in the animal. When the cardiac arrhythmia is attributable to re-entrant atrial flutter, the vector is preferably delivered locally to the right atrial myocardium of the animal (e.g. to the crista terminalis), and is more preferably delivered in a sustained-release manner. Delivery of the vector to a myocardial tissue can be effected by implanting a device (e.g. an implantable device comprising an electrode, such as a cardiac rhythm modulator or pacemaker) having a surface coated with a matrix comprising the vector in close proximity to the myocardial tissue. Preferably, the matrix is biodegradable and thereby delivers the vector to the tissue in a sustained-release manner.

The implantable device can be one which is made and used for the sole purpose of delivering the reverse gene therapy vector to the animal, or the device can be one which is applied to the surface of or inserted within the body of the animal for a purpose other than merely delivering the reverse gene therapy vector to the animal. By way of example, the implantable device can be a plurality of microspheres which comprise the reverse gene therapy vector and which are implanted into the body of the animal for the sole purpose of delivering the vector to the animal. Further by way of example, the implantable device can be a pacemaker having a surface coated with a matrix comprising the reverse gene therapy vector; the pacemaker is implanted in the vicinity of the animal's heart, both to modulate the animal's heartbeat when necessary and to deliver the vector to a cardiac tissue or to another tissue in close proximity to or in fluid communication with the coated surface of the pacemaker.

The reverse gene therapy vector can be incorporated into a coating of virtually any medical device. The coated devices provide a convenient means for local administration of the vector. For example, the vector can be incorporated into coatings for degradable and non-degradable sutures, orthopedic prostheses such as supporting rod implants, joint prostheses, pins for stabilizing fractures, bone cements and ceramics, tendon reconstruction implants, prosthetic implants, cardiovascular implants such as heart valve prostheses, pacemaker components, defibrillator components, angioplasty devices, intravascular stents, acute and in-dwelling catheters, ductus arteriosus closure devices, implants deliverable by cardiac catheters such as atrial and ventricular septal defect closure devices, urologic implants such as urinary catheters and stents, neurosurgical implants such as neurosurgical shunts, ophthalmologic implants such as lens prosthesis, thin ophthalmic sutures, and corneal implants, dental prostheses, internal and external wound dressings such as bandages and hernia repair meshes, pacemakers and other cardiac rhythm modulation devices, cardiac electrode leads, and other devices and implants, as will be apparent to the skilled artisan.

The reverse gene therapy compositions and methods described herein can be used to transforms cells located outside the body of the animal or cells located within the body of an animal. Following transformation of cells outside the body of the animal, the cells can be cultured, returned to the body of the same animal, or administered to the body of another animal of the same or different species, using substantially any known or subsequently developed method.

When the reverse gene therapy vector is delivered in the form of a particle which comprises the vector, the particle can be substantially any size. Preferably, the particle is a microparticle having a diameter less than about 900 micrometers, and preferably less than about 500 micrometers. Even more preferably, the particle is a nanoparticle having a diameter less than about 1 micrometer, and preferably less than about 600 nanometers. The vector can be present only on the surface of the particles, only at an interior portion of the particles, only in one or more layers of material in the particle, or throughout the particle. The particle preferably comprises a biocompatible material, and more preferably comprises a biodegradable material such as a polylactate-polyglycolate copolymer. Of course, substantially any known biocompatible polymeric or non-polymeric material can be used to form the particles, so long as at least a portion of the vector in or on the particle can be taken up by a cell which contacts the particle or is in fluid communication with the particle.

Cellular uptake of the gene therapy vector described herein can be enhanced by incorporating a specific cell surface receptor protein into the vector (e.g. fibroblast growth factor (FGF) or transferrin). Intracellular processing of the plasmid DNA within a lysosomal or endosomal compartment within the cell can be modulated by incorporating a lysosomotropic agent (e.g. sucrose or chloroquine) in order to reduce intracellular nuclease-mediated hydrolysis of the nucleic acid of the vector.

The reverse gene therapy vector preferably comprises a condensing agent. Condensation of DNA using polycations such as polylysine has also been demonstrated to enhance plasmid transfection by facilitating cell entry, possibly by encouraging nanoparticulate formation and protecting the DNA from nuclease mediated hydrolysis both extracellularly and within intracellular lysosomal or endosomal compartments. A preferred condensing agent is the polycation, polylysine.

The chemical identity of the condensing agent is not critical. The ability of a condensing agent to condense DNA or another nucleic acid or nucleic analog can be assessed using numerous methods known in the art. Effective amounts of such condensing agents can similarly be determined using these methods. For example, DNA condensation can be measured by comparing the kinetics in solution of condensed DNA and uncondensed DNA, and then further comparing the kinetics in the presence of a surfactant such as a detergent. It can also be measured by changes in the surface .zeta.-potential of the DNA in solution (Wolfert et al., 1996, Human Gene Therapy 7:2123-33), or by visualizing the DNA using an electron microscope (Laemmli, 1975, Proc. Natl. Acad. Sci. USA 72:4288-4292) or an atomic force microscope (Wolfert et al., 1996, Gene Therapy 3:269-273).

One preferred family of condensing agents is the polylysines. Polylysines are polypeptides of varying lengths, comprising (e.g. primarily or exclusively) lysine residues, which are positively charged at human physiological blood pH. The lysine residues can be D-lysine residues, L-lysine residues, or a mixture of the two enantiomers; poly-L-lysine is preferred. Polylysine has been demonstrated to be an efficacious DNA condensing agent (Laemmli, 1975, Proc. Natl. Acad. Sci. USA 72:4288-4292; Wolfert et al., 1996, Gene Therapy 3:269-273). The polylysines which are useful as condensing agents in the compositions and methods described herein include all variants of polylysine, regardless of length, linear, branched, or cross-linked structure, conformation, isomerization, or chemical modification, that are capable of condensing DNA or other polyanionic bioactive agents. Exemplary chemical modifications include methylation (Bello et al., 1985, J. Biomol. Struct. Dyn. 2:899-913) and glycosylation (Martinez-Fong et al., 1994, Hepatology 20:1602-1608). Such modifications can be made before or after synthesis of the polylysine. Other condensing agents which can be used to condense DNA and other nucleic acids include elemental cations, particularly divalent cations such as Mg2+ or Ca2+. Such cations can, for example, be used in the form of salts, such as MgCl2 or CaCl2. Other suitable elemental cations include Co3+ (particularly in the form of cobalt hexamine, Co(NH3)63+, or cobalt pentamine). La3+, Al3+, Ba2+, and Cs+. These cations are generally used in the form of a salt, particularly halide salts such as chloride and bromide salts, but other salts can be used as well.

It is understood that the ordinarily skilled physician or veterinarian will determine and prescribe an effective amount of the compound to alleviate the disease or disorder in the subject. In so proceeding, the physician or veterinarian can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. It is further understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the severity of the disease or disorder to be alleviated.

The invention encompasses the preparation and use of pharmaceutical compositions comprising the reverse gene therapy vector as an active ingredient. Such a pharmaceutical composition can consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition can comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. Administration of one of these pharmaceutical compositions to a subject is useful for alleviating a disease or disorder in the subject, as described elsewhere in the present disclosure.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient can be combined and which, following the combination, can be used to administer the active ingredient to a subject.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which arc suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.

Pharmaceutical compositions that are useful in the methods described herein can be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, and immunologically-based formulations.

A pharmaceutical composition can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition can comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition can further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include condensing agents such as polylysine.

Controlled- or sustained-release formulations of a pharmaceutical composition can be made using conventional technology.

A formulation of a pharmaceutical composition suitable for oral administration can be prepared, packaged, or sold in the form of a discrete solid dose unit including a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include a powdered or granular formulation, an aqueous or oily suspension, or an emulsion.

As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.

A tablet comprising the active ingredient can, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets can be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets can be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include potato starch and sodium starch glycolate. Known surface active agents include sodium lauryl sulfate. Known diluents include calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include corn starch and alginic acid. Known binding agents include gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include magnesium stearate, stearic acid, silica, and talc.

Tablets can be non-coated or they can be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate can be used to coat tablets. Further by way of example, tablets can be coated using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets can further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient can be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and can further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient can be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which can be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition which are suitable for oral administration can be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions can be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions can further comprise one or more additional ingredients including suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions can further comprise a thickening agent. Known suspending agents include sorbitol syrup, hydrogenated edible fats, sodium alginate. polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include lecithin and acacia. Known preservatives include methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

Powdered and granular formulations of a pharmaceutical preparation can be prepared using known methods. Such formulations can be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension by addition of an aqueous or oily vehicle thereto. Each of these formulations can further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, can also be included in these formulations.

A pharmaceutical composition can also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase can be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions can further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions can also contain additional ingredients including, for example, sweetening or flavoring agents.

A pharmaceutical composition can be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition can be in the form of, for example, a suppository, a retention enema preparation, and a suspension for rectal or colonic irrigation.

Suppository formulations can be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20oC.) and which is liquid at the rectal temperature of the subject (i.e. about 37oC. in a healthy human). Suitable pharmaceutically acceptable excipients include cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations can further comprise various additional ingredients including antioxidants and preservatives.

Retention enema preparations or suspensions for rectal or colonic irrigation can be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is known in the art, enema preparations can be administered using, and can be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations can further comprise various additional ingredients including antioxidants and preservatives.

A pharmaceutical composition can be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition can be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a suspension for vaginal irrigation.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily suspension into an absorbent material, with or without subsequent drying.

Douche preparations or suspensions for vaginal irrigation can be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is known in the art, douche preparations can be administered using, and can be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations can further comprise various additional ingredients including antioxidants, antibiotics, antifungal agents, and preservatives.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection and intravenous, intraarterial, or kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include suspensions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations can further comprise one or more additional ingredients including suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension. This suspension can be formulated according to the known art, and can comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation can comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and suspensions. Topically-administrable formulations can, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration can further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition can be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation can comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65oF. at atmospheric pressure. Generally the propellant can constitute 50 to 99.9% (w/w) of the composition, and the active ingredient can constitute 0.1 to 20% (w/w) of the composition. The propellant can further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery can also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations can be prepared, packaged, or sold as aqueous or dilute alcoholic suspensions, optionally sterile, comprising the active ingredient, and can conveniently be administered using any nebulization or atomization device. Such formulations can further comprise one or more additional ingredients including a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition.

Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration can, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and can further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition can be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations can, for example, be in the form of tablets or lozenges made using conventional methods, and can, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration can comprise a powder or an aerosolized or atomized suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and can further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition can be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations can, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops can further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

As used herein, "additional ingredients" include one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which can be included in the pharmaceutical compositions are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

Claim 1 of 13 Claims

What is claimed is:

1. A method of alleviating reentry atrial flutter in an affected animal cell, said method comprising locally delivering to a cardiac cell a reverse gene therapy vector comprising a promoter operably linked with a nucleic acid encoding a therapeutic gene product which is usually only expressed in cells of an abnormal tissue that is not afflicted with reentry atrial flutter, wherein said therapeutic gene product is a defective HERG protein, and delivery of said vector to the affected cardiac cell alleviates the flutter.


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